U.S. patent number 10,584,321 [Application Number 15/550,452] was granted by the patent office on 2020-03-10 for compositions and methods for transient delivery of nucleases.
This patent grant is currently assigned to University of Massachusetts. The grantee listed for this patent is University of Massachusetts. Invention is credited to Guangping Gao, Dan Wang, Phillip D. Zamore.
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United States Patent |
10,584,321 |
Gao , et al. |
March 10, 2020 |
Compositions and methods for transient delivery of nucleases
Abstract
The disclosure in some aspects relates to recombinant
adeno-associated viruses having nuclease grafted to one or more
capsid proteins. In some aspects, the disclosure relates to
isolated AAV capsid proteins having terminally grafted nucleases
and isolated nucleic acids encoding the same. Recent approaches to
delivering nucleases to cells for gene editing have focused on
delivering of expression vectors engineered to express the
nucleases in target cells. However, these approaches have proved to
be problematic in many instances due to genotoxicity resulting from
to prolonged expression of gene editing system in vivo. To prevent
such off-target genotoxicity due to prolonged presence of a gene
editing system, several studies explored delivery of mRNA or
protein instead of delivering the gene coding for the nucleases in
cell culture.
Inventors: |
Gao; Guangping (Westborough,
MA), Zamore; Phillip D. (Northborough, MA), Wang; Dan
(Belchertown, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
University of Massachusetts |
Boston |
MA |
US |
|
|
Assignee: |
University of Massachusetts
(Boston, MA)
|
Family
ID: |
56614963 |
Appl.
No.: |
15/550,452 |
Filed: |
February 12, 2016 |
PCT
Filed: |
February 12, 2016 |
PCT No.: |
PCT/US2016/017886 |
371(c)(1),(2),(4) Date: |
August 11, 2017 |
PCT
Pub. No.: |
WO2016/131009 |
PCT
Pub. Date: |
August 18, 2016 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180037877 A1 |
Feb 8, 2018 |
|
US 20180179501 A9 |
Jun 28, 2018 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
62115928 |
Feb 13, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
48/005 (20130101); C12N 7/00 (20130101); C12N
9/22 (20130101); C12N 15/11 (20130101); C07K
14/005 (20130101); C12N 15/907 (20130101); C12N
2750/14142 (20130101); C12N 2310/20 (20170501); C12N
2750/14122 (20130101); C07K 2319/735 (20130101); C12N
2750/14121 (20130101); A61K 38/00 (20130101); C12N
2750/14143 (20130101) |
Current International
Class: |
C12N
9/22 (20060101); C07K 14/005 (20060101); C12N
7/00 (20060101); C12N 15/11 (20060101); C12N
15/90 (20060101); A61K 48/00 (20060101); A61K
38/00 (20060101) |
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|
Primary Examiner: Ton; Thaian N.
Attorney, Agent or Firm: Wolf, Greenfield & Sacks,
P.C.
Parent Case Text
RELATED APPLICATIONS
This application is a National Stage Application of
PCT/US2016/017886, filed Feb. 12, 2016, entitled "COMPOSITIONS AND
METHODS FOR TRANSIENT DELIVERY OF NUCLEASES", which claims the
benefit under 35 U.S.C. .sctn. 119(e) of U.S. Provisional
Application Ser. No. 62/115,928, entitled "COMPOSITIONS AND METHODS
FOR TRANSIENT DELIVERY OF NUCLEASES" filed on Feb. 13, 2015, the
entire contents of each application which are incorporated herein
by reference.
Claims
What is claimed is:
1. An adeno-associated virus (AAV) capsid protein having a
terminally grafted nuclease.
2. The AAV capsid protein of claim 1, wherein the capsid protein is
a VP2 capsid protein.
3. The AAV capsid protein of claim 2, wherein the terminally
grafted nuclease is grafted to the N-terminus of the VP2 capsid
protein, or wherein the terminally grafted nuclease is grafted to
the C-terminus of the VP2 capsid protein.
4. The AAV capsid protein of claim 1, wherein the nuclease is
selected from: Transcription Activator-like Effector Nucleases
(TALENs), Zinc Finger Nucleases (ZFNs), engineered meganuclease,
re-engineered homing endonucleases and a Cas-family nuclease.
5. The AAV capsid protein of claim 2, further comprising a linker
conjugated to the C-terminus of the terminally grafted nuclease and
the N-terminus of the VP2 protein, or further comprising a linker
conjugated to the N-terminus of the terminally grafted nuclease and
the C-terminus of the VP2 protein.
6. The AAV capsid protein of claim 1, wherein the AAV capsid
protein having a terminally grafted nuclease is of: (i) an AAV1,
AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or
AAV12 serotype; or (ii) a serotype derived from a non-human
primate.
7. A recombinant adeno-associated virus (rAAV) comprising the
capsid protein of claim 1.
8. The rAAV of claim 7, wherein the rAAV comprises a transgene
encoding a guide RNA.
9. The rAAV of claim 7, wherein the AAV is an empty viral particle
with no transgene.
10. An in vitro method of targeting genome editing in a cell, the
method comprising: delivering to the cell a first recombinant adeno
associated virus (rAAV) having a terminally-grafted Cas-family
endonuclease on at least one capsid protein, wherein the
terminally-grafted Cas-family endonuclease is directed to a genomic
cleavage site by a guide RNA.
11. The method of claim 10, wherein the first rAAV comprises a
transgene encoding the guide RNA.
12. The method of claim 10 further comprising administering a
second rAAV having a transgene encoding a guide RNA that directs
the Cas-family endonuclease to a cleavage site in a target nucleic
acid.
13. A composition comprising: (i) a first recombinant
adeno-associated virus (rAAV) having an terminally-grafted nuclease
on at least one capsid protein; and (ii) a second rAAV having a
transgene encoding a guide RNA that directs the nuclease to a
cleavage site in a target nucleic acid.
14. An adeno-associated virus (AAV) capsid protein having a
terminally grafted nuclease or fragment thereof, wherein the
nuclease or fragment thereof comprises a terminally grafted
intein.
15. The AAV capsid protein of claim 14, wherein the capsid protein
is a VP2 capsid protein, or wherein the intein is IntN or IntC.
16. The composition of claim 13, wherein the nuclease is selected
from: Transcription Activator-like Effector Nucleases (TALENs),
Zinc Finger Nucleases (ZFNs), engineered meganuclease,
re-engineered homing endonucleases and a Cas-family nuclease.
17. The composition of claim 13, wherein the capsid protein is a
VP2 capsid protein.
18. The AAV capsid protein of claim 14, wherein the nuclease is
selected from: Transcription Activator-like Effector Nucleases
(TALENs), Zinc Finger Nucleases (ZFNs), engineered meganuclease,
re-engineered homing endonucleases and a Cas-family nuclease.
Description
FIELD OF THE INVENTION
The disclosure in some aspects relates to isolated nucleic acids,
compositions, and kits useful for protein delivery to cells.
BACKGROUND
Recently, gene editing using designer DNA sequence-specific
nucleases emerged as a technology for both basic biomedical
research and therapeutic development. Platforms based on three
distinct types of endonucleases have been developed for gene
editing, namely the zinc finger nuclease (ZFN), the transcription
activator-like effector nuclease (TALEN), and the clustered
regularly interspaced short palindromic repeat (CRISPR) associated
endonuclease 9 (cas9). Each nuclease is capable of inducing a DNA
double-stranded break (DSB) at specific DNA loci, thus triggering
two DNA repair pathways. The non-homologous end joining (NHEJ)
pathway generates random insertion/deletion (indel) mutations at
the DSB, whereas the homology-directed repair (HDR) pathway repairs
the DSB with the genetic information carried on a donor template.
Therefore, these gene editing platforms are capable of manipulating
genes at specific genomic loci in multiple ways, such as disrupting
gene function, repairing a mutant gene to normal, and inserting DNA
material.
Transforming the gene editing technology into therapeutic uses
encounters several obstacles, including the concern over safety.
Certain gene editing platforms have been shown to induce off-target
DSBs throughout genomes, which is associated with genotoxicity.
Such off-target effects not only stem from the intrinsic ambiguity
of DNA sequence recognition by nucleases, but also attribute to the
prolonged presence of an active gene editing system in a given
cell. As a result, off-target DSBs accumulate over time, and
ultimately lead to genotoxicity.
SUMMARY
Recent approaches to delivering nucleases to cells for gene editing
have focused on delivering of expression vectors engineered to
express the nucleases in target cells. However, these approaches
have proved to be problematic in many instances due to genotoxicity
resulting from to prolonged expression of gene editing system in
vivo. To prevent such off-target genotoxicity due to prolonged
presence of a gene editing system, several studies explored
delivery of mRNA or protein instead of delivering the gene coding
for the nucleases in cell culture. As a result, the gene editing
system functions only in a short period of time until the nuclease
mRNA or protein is naturally degraded inside cells, which has been
shown to reduce off-target effects. However, delivery of mRNA or
protein in vivo is a significant task, and the delivery efficiency
is very limited with conventional techniques. In contrast, the
present disclosure overcomes such genotoxicity and delivery issues
by using viruses for transiently delivering nucleases to cells
thereby fulfilling the task of inducing permanent gene editing in a
transient manner such that the nucleases will degrade naturally. In
some embodiments, the disclosure relates to the uses of a viral
vector (e.g., an AAV) as a delivery vehicle to carry a nuclease
(e.g., a Cas9 protein or other designer nuclease proteins) to
cells. In some embodiments, to avoid the potential genotoxicity due
to prolonged expression of gene editing system in vivo, methods are
provided herein to transiently deliver an endonuclease protein
using recombinant adeno-associated viruses. In some embodiments,
AAV capsid is used as a delivery vehicle to carry the Cas9 protein
or other designer nuclease proteins.
In some aspects, the disclosure relates to an adeno-associated
virus (AAV) capsid protein having a terminally grafted
nuclease.
In some embodiments, the capsid protein is a VP2 capsid protein. In
some embodiments, the terminally grafted nuclease is grafted to the
N-terminus of the VP2 capsid protein. In some embodiments, the
terminally grafted nuclease is grafted to the C-terminus of the VP2
capsid protein.
In some embodiments, the nuclease is selected from: Transcription
Activator-like Effector Nucleases (TALENs), Zinc Finger Nucleases
(ZFNs), engineered meganuclease, re-engineered homing endonucleases
and a Cas-family nuclease. In some embodiments, the nuclease is a
Cas-family nuclease selected from the group consisting of Cas9 and
Cas7. In some embodiments, the nuclease is represented by SEQ ID
NO: 2. In some embodiments, the nuclease is a polypeptide encoded
by the nucleic acid sequence represented by SEQ ID NO: 1.
In some embodiments, the AAV capsid protein further comprises a
linker conjugated to the C-terminus of the terminally grafted
nuclease and the N-terminus of the VP2 protein. In some
embodiments, the AAV capsid protein further comprises a linker
conjugated to the N-terminus of the terminally grafted nuclease and
the C-terminus of the VP2 protein.
In some embodiments, the AAV capsid protein hays an terminally
grafted nuclease is of a serotype derived from a non-human primate.
In some embodiments, the AAV capsid protein has an terminally
grafted nuclease is selected from: AAV1, AAV2, AAV3, AAV4, AAV5,
AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.
In some aspects, the disclosure relates to a recombinant
adeno-associated virus (rAAV) comprising an adeno-associated virus
(AAV) capsid protein having a terminally grafted nuclease.
In some embodiments, the rAAV comprises a transgene. In some
embodiments, the transgene encodes a guide RNA. In some
embodiments, the guide RNA directs the nuclease to a cleavage site
in a target nucleic acid.
In some embodiments, the AAV is an empty viral particle with no
transgene.
In some aspects, the disclosure provides a composition comprising
an rAAV as described by this document. In some embodiments, the
composition further comprises a pharmaceutically acceptable
carrier.
In some aspects, the disclosure relates to a nucleic acid encoding
an AAV capsid protein having an terminally grafted nuclease. In
some embodiments, a host cell contains the nucleic acid. In some
embodiments, the host cell contains a nucleic acid encodes an AAV
VP2 capsid protein having an terminally grafted nuclease. In some
embodiments, the host cell further comprises one or more nucleic
acids encoding VP1 and VP3 capsid proteins.
In some aspects, the disclosure relates to a composition comprising
a host cell as described by this document and a sterile cell
culture medium. In some aspects, the disclosure relates to a
composition comprising a host cell as described by this document
and a cryopreservative.
In some aspects, the disclosure relates to an isolated nucleic acid
comprising a sequence represented by SEQ ID NO: 3.
In some aspects, the disclosure relates to an isolated nucleic acid
encoding an AAV capsid protein having an amino acid sequence
selected from the group consisting of: SEQ ID NOs: 2 and 4.
In some aspects, the disclosure relates to an isolated AAV capsid
protein comprising an amino acid sequence selected from the group
consisting of: SEQ ID NOs: 2 and 4.
In some aspects, the disclosure relates to a composition comprising
an isolated AAV capsid protein as described by this document. In
some embodiments, the composition further comprises a
pharmaceutically acceptable carrier.
In some aspects, the disclosure relates to a kit for producing a
rAAV, the kit comprising: a container housing an isolated nucleic
acid having a sequence of SEQ ID NO: 1 or 3. In some embodiments,
the kit further comprises instructions for producing the rAAV. In
some embodiments, the kit further comprises at least one container
housing a recombinant AAV vector, wherein the recombinant AAV
vector comprises a transgene.
In some aspects, the disclosure relates to a kit comprising: a
container housing a recombinant AAV having an isolated AAV capsid
protein having an amino acid sequence as set forth in SEQ ID NO: 2
or 4.
In some aspects, the disclosure relates to a method of targeting
genome editing in a cell, the method comprising: delivering to the
cell a first recombinant adeno associated virus (rAAV) having an
terminally-grafted nuclease on at least one capsid protein, wherein
when present in the cell, the terminally-grafted nuclease is
directed to a genomic cleavage site by a guide RNA.
In some embodiments of the method, the first rAAV comprises a
transgene encoding the guide RNA.
In some embodiments, the method further comprises administering a
second rAAV having a transgene encoding a guide RNA that directs
the nuclease to a cleavage site in a target nucleic acid.
In some embodiments, the cell is present in a subject, and the
first rAAV or second rAAV is administered to the subject
intravenously, intravascularly, transdermally, intraocularly,
intrathecally, orally, intramuscularly, subcutaneously,
intranasally, or by inhalation, thereby delivering the first rAAV
or second rAAV to the cell. In some embodiments, the subject is
selected from a mouse, a rat, a rabbit, a dog, a cat, a sheep, a
pig, and a non-human primate. In some embodiments, the subject is a
human.
In some aspects, the disclosure relates to a composition
comprising: i.) a first recombinant adeno-associated virus (rAAV)
having an terminally-grafted nuclease on at least one capsid
protein; and ii.) a second rAAV having a transgene encoding a guide
RNA that directs the nuclease to a cleavage site in a target
nucleic acid.
In some embodiments, the first rAAV is an empty viral particle. In
some embodiments, the first rAAV has an terminally-grafted nuclease
that is grafted to the C-terminus of a VP2 capsid protein of the
rAAV.
In some aspects, the disclosure relates to an adeno-associated
virus (AAV) capsid protein having a terminally grafted nuclease or
fragment thereof, wherein the nuclease or fragment thereof
comprises a terminally grafted intein.
In some embodiments, the capsid protein is a VP2 capsid protein. In
some embodiments, the intein is IntN or IntC. In some embodiments,
the capsid protein is represented by any one of SEQ ID NO: 7 to
9.
Each of the limitations of the disclosure can encompass various
embodiments of the disclosure. It is, therefore, anticipated that
each of the limitations of the disclosure involving any one element
or combinations of elements can be included in each aspect of the
disclosure. This disclosure is not limited in its application to
the details of construction and the arrangement of components set
forth in the following description or illustrated in the drawings.
The disclosure is capable of other embodiments and of being
practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF DRAWINGS
The accompanying drawings are not intended to be drawn to scale. In
the drawings, each identical or nearly identical component that is
illustrated in various figures is represented by a like numeral.
For purposes of clarity, not every component may be labeled in
every drawing. In the drawings:
FIGS. 1A-1B shows the SpCas9-VP2 fusion protein is produced in
HEK293 cells. FIG. 1A shows the HA tagged SpCas9 is fused to the
N-terminus of VP2. The expression of this fusion protein is driven
by the CMV promoter. BGHpA: bovine growth hormone polyadenylation
signal. FIG. 1B depicts western blotting using anti-HA antibody
showing the HA-tagged fusion protein (.about.230 kD, arrow)
produced from transiently transfected HEK293 cells. HA tagged
SpCas9 (.about.162 kD) is marked by the triangle. Star indicates a
band of unknown origin, a likely degradation product from the
fusion protein.
FIGS. 2A-2B show the SpCas9-VP2 fusion protein mediates gene
editing in HEK293 cells. FIG. 2A shows the DNA repair reporter
construct. The mutant GFP (GFPmut) carries a disruptive insertion
(Ins), followed by out-of-frame (+3 frame) T2A and mCherry. In the
presence of a functional gene editing system targeting Ins, +1
insertion by NHEJ shifts the T2A and mCherry to in-frame, resulting
mCherry fluorescence. FIG. 2B shows the results of a reporter assay
in HEK293 cells by co-transfection of the reporter construct and
various plasmid as indicated. Both mCherry fluorescence and bright
field images are shown. Scale bar=50 .mu.M.
FIGS. 3A-3B show alternative strategies utilizing intein-mediated
protein trans-splicing (PTS). FIG. 3A shows the N-terminus and
C-terminus Npu DnaE intein (IntN and IntC,) are fused with SpCas9
and VP2, respectively. The IntC-VP2 is packaged into AAV virion.
PTS occurs between the SpCas9-IntN fusion protein and the IntC-AAV
chimeric virion to produce the SpCas9-AAV virion. FIG. 3B shows
that in the first AAV vector, the AAV genome encodes the N-terminal
portion of SpCas9 (SpCas9N) fused with IntN. The second AAV vector
carries IntC and the C-terminal portion of SpCas9 fused to VP2. In
vivo transduction of the first AAV vector produces the fusion
protein SpCas9N-IntN, which is followed by delivery of the second
vector. PTS occurs to reconstitute the full-length SpCas9
protein.
FIG. 4 shows an expression construct comprising a nucleic acid
sequence encoding SpCas9 nuclease N-terminally fused to VP2 capsid
protein.
FIG. 5 shows co-transfection of Split Cas9 parts in HEK293 cells
reconstituted SpCas9 and VP2 fusion protein, as measured by Western
blot. Ctrl: pCMV-SpCas9-(EAAAKx3)-VP2; N:
pU1a-Cas9.sub.n-Int.sub.n; C part: Int.sub.cCas9.sub.c-( )-VP2; HA
tag is present in SpCas9 N-terminal. The designation "( )" refers
to a linker sequence (e.g., GS, GGGGSx3, EAAAKx3).
FIG. 6 shows co-transfection of Split Cas9 parts in HEK293 cells
reconstituted gene editing function. Cells were transfected with
EGFP-ON reporter, pU1a-Cas9.sub.n-Int.sub.n, and
Int.sub.c-Cas9.sub.C-( )-VP2. EGFP reports Cas9 cleavage and NHEJ
repair; mCherry is constitutively expressed as control.
FIG. 7 shows incorporation of Int.sub.C-SpCas9.sub.c polypeptide
onto rAAV2 capsid. Cells were transfected with plasmid encoding VP1
and VP3 proteins, and a plasmid encoding Int.sub.c-SpCas9.sub.c-(
)-VP2. Purified rAAV particles were examined by silver
staining.
DETAILED DESCRIPTION
Genome editing is a powerful tool for the interrogation and
manipulation of biological functions within cells. For example,
genome editing allows for the repair of mutant genes to normal
function, disruption of gene function and the insertion of genetic
material (e.g. DNA), all at specific genomic loci. However, several
challenges associated with the delivery and prolonged expression of
nucleases in cells, such as genotoxicity due to off-target cleavage
of DNA, has limited the therapeutic effectiveness of gene editing
platforms. The instant disclosure overcomes current limitations by
providing compositions and methods that improve delivery of genome
editing nucleases. Accordingly, in some aspects, the disclosure
relates to viral proteins comprising a terminally grafted
nucleases.
As used herein, "genome editing" refers to adding, disrupting or
changing genomic sequences (e.g., a gene sequence). In some
embodiments, genome editing is performed using engineered proteins
and related molecules. In some aspects, genome editing comprises
the use of engineered nucleases to cleave a target genomic locus.
In some embodiments, genome editing further comprises inserting,
deleting, mutating or substituting nucleic acid residues at a
cleaved locus. In some embodiments, inserting, deleting, mutating
or substituting nucleic acid residues at a cleaved locus is
accomplished through endogenous cellular mechanisms such as
homologous recombination (HR) and non-homologous end joining
(NHEJ). Exemplary genome editing technologies include, but are not
limited to Transcription Activator-like Effector Nucleases
(TALENs), Zinc Finger Nucleases (ZFNs), engineered meganuclease
re-engineered homing endonucleases, the CRISPR/Cas system. In some
embodiments, the gene editing technologies are proteins or
molecules related to TALENs, including but not limited to
transcription activator-like effectors (TALEs) and restriction
endonucleases (e.g. FokI). In some embodiments, the gene editing
technologies are proteins or molecules related to ZFNs, including
but not limited to proteins comprising the Cys.sub.2His.sub.2 fold
group (for example Zif268 (EGR1)), and restriction endonucleases
(e.g. FokI). In some embodiments, the gene editing technologies are
proteins or molecules related to the CRISPR/Cas system, including
but not limited to Cas9, Cas6, dCas9, CRISPR RNA (crRNA) and
trans-activating crRNA (tracrRNA).
As used herein, the terms "endonuclease" and "nuclease" refer to an
enzyme that cleaves a phosphodiester bond or bonds within a
polynucleotide chain. Nucleases may be naturally occurring or
genetically engineered. Genetically engineered nucleases are
particularly useful for genome editing and are generally classified
into four families: zinc finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs), engineered
meganucleases and CRISPR-associated proteins (Cas nucleases). In
some embodiments, the nuclease is a ZFN. In some embodiments, the
ZFN comprises a FokI cleavage domain. In some embodiments, the ZFN
comprises Cys.sub.2His.sub.2 fold group. In some embodiments, the
nuclease is a TALEN. In some embodiments, the TALEN comprises a
FokI cleavage domain. In some embodiments, the nuclease is an
engineered meganuclease.
The term "CRISPR" refers to "clustered regularly interspaced short
palindromic repeats", which are DNA loci containing short
repetitions of base sequences. CRISPR loci form a portion of a
prokaryotic adaptive immune system that confers resistance to
foreign genetic material. Each CRISPR loci is flanked by short
segments of "spacer DNA", which are derived from viral genomic
material. In the Type II CRISPR system, spacer DNA hybridizes to
transactivating RNA (tracrRNA) and is processed into CRISPR-RNA
(crRNA) and subsequently associates with CRISPR-associated
nucleases (Cas nucleases) to form complexes that recognize and
degrade foreign DNA. In certain embodiments, the nuclease is a
CRISPR-associated nuclease (Cas nuclease). Examples of CRISPR
nucleases include, but are not limited to Cas9, Cas6 and dCas9.
dCas9 is an engineered Cas protein that binds to a target locus but
does not cleave said locus. In some embodiments, the nuclease is
Cas9. In some embodiments, the Cas9 is derived from the bacteria S.
pyogenes (SpCas9).
For the purpose of genome editing, the CRISPR system can be
modified to combine the tracrRNA and crRNA in to a single guide RNA
(sgRNA) or just (gRNA). As used herein, the term "guide RNA" or
"gRNA" refers to a polynucleotide sequence that is complementary to
a target sequence in a cell and associates with a Cas nuclease,
thereby directing the Cas nuclease to the target sequence. In some
embodiments, a gRNA ranges between 1 and 30 nucleotides in length.
In some embodiments, a gRNA ranges between 5 and 25 nucleotides in
length. In some embodiments, a gRNA ranges between 10 and 20
nucleotides in length. In some embodiments, a gRNA ranges between
14 and 18 nucleotides in length.
Aspects of the disclosure relate to SpCas9 grafted to an AAV2
capsid protein, VP2. However, in some embodiments, the same
strategy can be applied in other contexts. For example, the SpCas9
can be replaced with any modified SpCas9 such as mutated or
truncated forms, Cas9 proteins from other species and nucleases
used in other gene editing platforms such as ZFNs and TALENs. In
some embodiments, a nuclease terminally grafted to an AAV2 capsid
protein may also be fused to another functional domain, for example
single guide RNA (sgRNA).
Similarly, the AAV2 capsid protein VP2 may be replaced with VP2 of
other AAV serotypes (e.g., AAV3, AAV3b, AAV4, AAV5, AAV6, AAV7,
AAV8, AAV9, AAVrh8, AAV10, and variants thereof), or a suitable
capsid protein of any viral vector. Thus, in some aspects, the
disclosure relates to the viral delivery of a nuclease. Examples of
viral vectors include retroviral vectors (e.g. Maloney murine
leukemia virus, MML-V), adenoviral vectors (e.g. AD100), lentiviral
vectors (HIV and FIV-based vectors), herpesvirus vectors (e.g.
HSV-2). In some embodiments, the disclosure relates to
adeno-associated viruses (AAVs). In some embodiments, a nuclease is
grafted to or replaces all or a portion of a viral
glycoprotein.
In some embodiments, SpCas-VP2 is incorporated into AAV2 capsid to
form AAV virion. In some embodiments, the start codon of VP2 is
mutated in the cap gene from the trans AAV production plasmid. In
some embodiments, when Cas9 is fused to the N-terminus of VP2, the
resulting Cas9-VP2 fusion protein is functional with respect to
both productive AAV assembly and being an active component of the
CRISPR/Cas9 gene editing system.
In some embodiments, a catalytically deficient form of the cas9
protein (dCas9) is fused with a C-terminal peptide domain that
either activates or represses gene expression. In such embodiments,
such a dCas9-effector fusion protein binds DNA in a sgRNA-guided
manner.
In some aspects, the disclosure relates to the discovery that
inteins can be utilized to rejoin (e.g., reconstitute) fragments or
portions of gene editing proteins to generate a functional gene
editing protein that is grafted onto an AAV capsid protein. As used
herein, "intein" refers to a self-splicing protein intron (e.g.,
peptide) that ligates flanking N-terminal and C-terminal exteins
(e.g., fragments to be joined). The use of certain inteins for
joining heterologous protein fragments is described, for example,
in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014). For
example, when fused to separate protein fragments, the inteins IntN
and IntC recognize each other, splice themselves out and
simultaneously ligate the flanking N- and C-terminal exteins of the
protein fragments to which they were fused, thereby reconstituting
a full length protein from the two protein fragments. Other
suitable inteins will be apparent to a person of skill in the
art.
A nuclease protein fragment (e.g., Cas9 fragment) can vary in
length. In some embodiments, a protein fragment ranges from 2 amino
acids to about 1000 amino acids in length. In some embodiments, a
protein fragment ranges from about 5 amino acids to about 500 amino
acids in length. In some embodiments, a protein fragment ranges
from about 20 amino acids to about 200 amino acids in length. In
some embodiments, a protein fragment ranges from about 10 amino
acids to about 100 amino acids in length. Suitable protein
fragments of other lengths will be apparent to a person of skill in
the art.
In some embodiments, a portion or fragment of a nuclease (e.g., a
fragment of Cas9) is fused to an intein. The nuclease can be fused
to the N-terminus or the C-terminus of the intein. In some
embodiments, a portion or fragment of a nuclease (e.g., a fragment
of Cas9) is fused to an intein and fused to an AAV capsid protein.
The intein, nuclease and capsid protein can be fused together in
any arrangement (e.g., nuclease-intein-capsid,
intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some
embodiments, the N-terminus of an intein is fused to the C-terminus
of a nuclease (e.g., Cas9) and the C-terminus of the intein is
fused to the N-terminus of an AAV capsid protein.
In some embodiments, the IntN/IntC system is used to join fragments
of a nuclease. In some embodiments, IntC is fused to the N-terminus
of a nuclease (e.g., Cas9) fragment that is grafted to an AAV
capsid protein. In some embodiments, IntN is fused to the
C-terminus of a nuclease (e.g., Cas9) fragment. In some
embodiments, a fragment of a nuclease fused to an intein is
represented by SEQ ID NO: 6. In some embodiments, an AAV capsid
protein comprising an intein fused to a fragment of a nuclease that
has been terminally grafted to the AAV capsid protein is
represented by any one of SEQ ID NO: 7 to 9.
Isolated AAV Capsid Proteins and Nucleic Acids Encoding the
Same
AAVs disclosed herein are useful for creating vectors that
facilitate delivery of nucleases to cells for human gene editing
applications. Protein and amino acid sequences as well as other
information regarding the AAVs capsid are set forth in the sequence
listing.
In some embodiments, an AAV capsid having a terminally graft
nuclease is provided that has an amino acid sequence represented by
SEQ ID NO: 4. In some embodiments, an AAV capsid having a
terminally graft nuclease is provided that is encoded by a nucleic
acid sequence represented by SEQ ID NO: 3.
An example of an isolated nucleic acid that encodes an AAV capsid
protein having a terminally graft nuclease is a nucleic acid having
a sequence of: SEQ ID NO: 3 as well as nucleic acids having
substantial homology thereto. In some embodiments, isolated nucleic
acids that encode AAV capsids are provided that encode the VP2
protein portion of the amino acid sequence represented by SEQ ID
NO: 3.
In some embodiments, nucleic acids are provided that encode an AAV
capsid having a nuclease grafted within its capsid sequence (e.g.,
a AAV9 capsid) and up to 5, up to 10, up to 20, up to 30, up to 40,
up to 50, up to 100 other amino acid alternations.
In some embodiments, a fragment (portion) of an isolated nucleic
acid encoding a AAV capsid sequence may be useful for constructing
a nucleic acid encoding a desired capsid sequence. Fragments may be
of any appropriate length (e.g., at least 9, at least 18, at least
36, at least 72, at least 144, at least 288, at least 576, at least
1152 or more nucleotides in length). For example, a fragment of
nucleic acid sequence encoding a variant amino acid (compared with
a known AAV serotype) may be used to construct, or may be
incorporated within, a nucleic acid sequence encoding an AAV capsid
sequence to alter the properties of the AAV capsid. For example, a
nucleic sequence encoding an AAV variant may comprise n amino acid
variants (e.g., in which n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)
compared with a known AAV serotype (e.g., AAV9). A recombinant cap
sequence may be constructed having one or more of the n amino acid
variants by incorporating fragments of a nucleic acid sequence
comprising a region encoding a variant amino acid into the sequence
of a nucleic acid encoding the known AAV serotype. The fragments
may be incorporated by any appropriate method, including using site
directed mutagenesis. In some embodiments, polypeptide fragments
that are not normally present in AAV capsid proteins may be
incorporated into a recombinant cap sequence. In some embodiments,
the polypeptide fragment is grafted onto the recombinant cap
sequence. Thus, new AAV variants may be created having new
properties.
As used herein, "grafting" refers to joining or uniting of at least
two polymeric molecules. In some embodiments, the term grafting
refers joining or uniting of at least two polymeric molecules such
that one of the at least two molecules is inserted within another
of the at least two molecules. In some embodiments, the term
grafting refers to joining or uniting of at least two polymeric
molecules such that one of the at least two molecules is appended
to another of the at least two molecules. In some embodiments, the
term grafting refers joining or uniting of at least two nucleic
acid molecules such that one of the at least two nucleic acid
molecules is inserted within another of the at least two nucleic
acid molecules. In some embodiments, the term grafting refers to
joining or uniting of at least two nucleic acid molecules such that
one of the at least two molecules is appended to another of the at
least two nucleic acid molecules.
In some embodiments, a grafted nucleic acid molecule encodes a
chimeric protein. In some embodiments, a grafted nucleic acid
molecule encodes a chimeric protein, such that one polypeptide is
effectively inserted into another polypeptide (e.g. not directly
conjugated before the N-terminus or after the C-terminus), thereby
creating a contiguous fusion of two polypeptides. In some
embodiments, a grafted nucleic acid molecule encodes a chimeric
protein, such that one polypeptide is effectively appended to
another polypeptide (e.g. directly conjugated before the N-terminus
or after the C-terminus), thereby creating a contiguous fusion of
two polypeptides. In some embodiments, the term grafting refers to
joining or uniting of at least two polypeptides, or fragments
thereof, such that one of the at least two polypeptides or
fragments thereof is inserted within another of the at least two
polypeptides or fragments thereof. In some embodiments, the term
grafting refers to joining or uniting of at least two polypeptides
or fragments thereof such that one of the at least two polypeptides
or fragments thereof is appended to another of the at least two
polypeptides or fragments thereof.
In some embodiments, the instant disclosure relates to an
adeno-associated virus (AAV) capsid protein comprising a AAV capsid
protein having an N-terminally grafted nuclease.
In some embodiments, the AAV capsid protein further comprises a
linker. Non-limiting examples of linkers include flexible linkers
(e.g. glycine-rich linkers), rigid linkers (e.g. [EAAK].sub.n,
where n>2), and cleavable linkers (e.g. protease-sensitive
sequences). Other linkers are disclosed, for example in Chen et
al., Fusion protein linkers: Property, design and functionality.
Advanced drug delivery reviews, 2013. In some embodiments, the
linker is conjugated to the C-terminus of a terminally grafted
nuclease (e.g., an N-terminally grafted nuclease). In some
embodiments, the linker is conjugated to the N-terminus of the
terminally grafted nuclease (e.g., an N-terminally grafted
nuclease). In some embodiments, one linker is conjugated to the
N-terminus of the terminally grafted nuclease and a second linker
is conjugated to the C-terminus of the terminally grafted
nuclease.
In some embodiments, the linker is a glycine-rich linker. In some
embodiments, the linker comprises at least one polypeptide repeat,
each repeat comprising at least 80% glycine residues. In some
embodiments, the polypeptide repeat comprises GGGS (SEQ ID NO: 5).
In some embodiments, the linker comprises a formula selected from
the group consisting of: [G].sub.n, [G].sub.nS, [GS].sub.n, and
[GGSG].sub.n, wherein G is glycine and wherein n is an integer
greater than one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more).
In some cases, fragments of capsid proteins disclosed herein are
provided. Such fragments may at least 10, at least 20, at least 50,
at least 100, at least 200, at least 300, at least 400, at least
500 or more amino acids in length. In some embodiments, chimeric
capsid proteins are provided that comprise one or more fragments of
one or more capsid proteins disclosed herein.
"Homology" refers to the percent identity between two
polynucleotides or two polypeptide moieties. The term "substantial
homology", when referring to a nucleic acid, or fragment thereof,
indicates that, when optimally aligned with appropriate nucleotide
insertions or deletions with another nucleic acid (or its
complementary strand), there is nucleotide sequence identity in
about 90 to 100% of the aligned sequences. When referring to a
polypeptide, or fragment thereof, the term "substantial homology"
indicates that, when optimally aligned with appropriate gaps,
insertions or deletions with another polypeptide, there is
nucleotide sequence identity in about 90 to 100% of the aligned
sequences. The term "highly conserved" means at least 80% identity,
preferably at least 90% identity, and more preferably, over 97%
identity. In some cases, highly conserved may refer to 100%
identity. Identity is readily determined by one of skill in the art
by, for example, the use of algorithms and computer programs known
by those of skill in the art.
As described herein, alignments between sequences of nucleic acids
or polypeptides are performed using any of a variety of publicly or
commercially available Multiple Sequence Alignment Programs, such
as "Clustal W", accessible through Web Servers on the internet.
Alternatively, Vector NTI utilities may also be used. There are
also a number of algorithms known in the art which can be used to
measure nucleotide sequence identity, including those contained in
the programs described above. As another example, polynucleotide
sequences can be compared using BLASTN, which provides alignments
and percent sequence identity of the regions of the best overlap
between the query and search sequences. Similar programs are
available for the comparison of amino acid sequences, e.g., the
"Clustal X" program, BLASTP. Typically, any of these programs are
used at default settings, although one of skill in the art can
alter these settings as needed. Alternatively, one of skill in the
art can utilize another algorithm or computer program which
provides at least the level of identity or alignment as that
provided by the referenced algorithms and programs. Alignments may
be used to identify corresponding amino acids between two proteins
or peptides. A "corresponding amino acid" is an amino acid of a
protein or peptide sequence that has been aligned with an amino
acid of another protein or peptide sequence. Corresponding amino
acids may be identical or non-identical. A corresponding amino acid
that is a non-identical amino acid may be referred to as a variant
amino acid.
Alternatively for nucleic acids, homology can be determined by
hybridization of polynucleotides under conditions which form stable
duplexes between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art.
A "nucleic acid" sequence refers to a DNA or RNA sequence. In some
embodiments, proteins and nucleic acids of the disclosure are
isolated. As used herein, the term "isolated" means artificially
produced. As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art. As used
herein with respect to proteins or peptides, the term "isolated"
refers to a protein or peptide that has been isolated from its
natural environment or artificially produced (e.g., by chemical
synthesis, by recombinant DNA technology, etc.).
The skilled artisan will also realize that conservative amino acid
substitutions may be made to provide functionally equivalent
variants, or homologs of the capsid proteins. In some aspects the
disclosure embraces sequence alterations that result in
conservative amino acid substitutions. As used herein, a
conservative amino acid substitution refers to an amino acid
substitution that does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references that compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F.
M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Conservative substitutions of amino acids include substitutions
made among amino acids within the following groups: (a) M, I, L, V;
(b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E,
D. Therefore, one can make conservative amino acid substitutions to
the amino acid sequence of the proteins and polypeptides disclosed
herein.
Recombinant AAVs
In some aspects, the disclosure provides isolated AAVs. As used
herein with respect to AAVs, the term "isolated" refers to an AAV
that has been artificially produced or obtained. Isolated AAVs may
be produced using recombinant methods. Such AAVs are referred to
herein as "recombinant AAVs". Recombinant AAVs (rAAVs) preferably
have tissue-specific targeting capabilities, such that a nuclease
and/or transgene of the rAAV will be delivered specifically to one
or more predetermined tissue(s). The AAV capsid is an important
element in determining these tissue-specific targeting
capabilities. Thus, an rAAV having a capsid appropriate for the
tissue being targeted can be selected Methods for obtaining
recombinant AAVs having a desired capsid protein are well known in
the art. (See, for example, US 2003/0138772), the contents of which
are incorporated herein by reference in their entirety). Typically
the methods involve culturing a host cell which contains a nucleic
acid sequence encoding an AAV capsid protein; a functional rep
gene; a recombinant AAV vector composed of, AAV inverted terminal
repeats (ITRs) and a transgene; and sufficient helper functions to
permit packaging of the recombinant AAV vector into the AAV capsid
proteins. In some embodiments, capsid proteins are structural
proteins encoded by the cap gene of an AAV. AAVs comprise three
capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3),
all of which are transcribed from a single cap gene via alternative
splicing. In some embodiments, the molecular weights of VP1, VP2
and VP3 are respectively about 87 kDa, about 72 kDa and about 62
kDa. In some embodiments, upon translation, capsid proteins form a
spherical 60-mer protein shell around the viral genome. In some
embodiments, the functions of the capsid proteins are to protect
the viral genome, deliver the genome and interact with the host. In
some aspects, capsid proteins deliver the viral genome to a host in
a tissue specific manner. In some embodiments, the a terminally
grafted nuclease is present on all three capsid proteins (e.g. VP1,
VP2, VP3) of a rAAV. In some embodiments, the terminally grafted
nuclease is present on two of the capsid proteins (e.g. VP2 and
VP3) of a rAAV. In some embodiments, the terminally grafted
nuclease is present on a single capsid protein of a rAAV. In some
embodiments, the terminally grafted nuclease is present on the VP2
capsid protein of the rAAV.
In some embodiments, the instant disclosure relates to an
adeno-associated virus (AAV) capsid protein comprising: an AAV
capsid protein having an N-terminally grafted nuclease, wherein the
AAV capsid protein is not of an AAV2 serotype. In some embodiments,
the AAV capsid protein is of an AAV serotype selected from the
group consisting of AAV3, AAV4, AAV5, AAV6, AAV8, AAVrh8 AAV9, and
AAV10. In some embodiments, the capsid protein having an
N-terminally grafted nuclease is a viral protein 2 (VP2) capsid
protein. In some embodiments, the AAV capsid protein having a
terminally grafted nuclease is of a serotype derived from a
non-human primate. In some embodiments, the AAV capsid protein
having a terminally grafted nuclease is of a AAVrh8 serotype. In
some embodiments, the AAV capsid protein having an N-terminally
grafted nuclease is of an AAV9, optionally AAV9.47, serotype.
In some aspects, the instant disclosure relates to the location
within an AAV capsid protein where a nuclease is grafted. In some
embodiments, the nuclease is N-terminally grafted to the capsid
protein. In some embodiments, the nuclease is C-terminally grafted
to a capsid protein. In some embodiments, a nuclease that is
C-terminally grafted to a capsid protein (e.g., VP2) resides within
the viral particle, and the viral particle does not contain a
genome, e.g., a nucleic acid harboring a transgene.
The components to be cultured in the host cell to package a rAAV
vector in an AAV capsid may be provided to the host cell in trans.
Alternatively, any one or more of the required components (e.g.,
recombinant AAV vector, rep sequences, cap sequences, and/or helper
functions) may be provided by a stable host cell which has been
engineered to contain one or more of the required components using
methods known to those of skill in the art. Most suitably, such a
stable host cell will contain the required component(s) under the
control of an inducible promoter. However, the required
component(s) may be under the control of a constitutive promoter.
Examples of suitable inducible and constitutive promoters are
provided herein, in the discussion of regulatory elements suitable
for use with the transgene. In still another alternative, a
selected stable host cell may contain selected component(s) under
the control of a constitutive promoter and other selected
component(s) under the control of one or more inducible promoters.
For example, a stable host cell may be generated which is derived
from 293 cells (which contain E1 helper functions under the control
of a constitutive promoter), but which contain the rep and/or cap
proteins under the control of inducible promoters. Still other
stable host cells may be generated by one of skill in the art.
In some embodiments, the instant disclosure relates to a host cell
containing a nucleic acid that comprises a coding sequence encoding
a nuclease terminally grafted to a capsid protein that is operably
linked to a promoter. In some embodiments, the instant disclosure
relates to a composition comprising the host cell described above.
In some embodiments, the composition comprising the host cell above
further comprises a cryopreservative.
The recombinant AAV vector, rep sequences, cap sequences, and
helper functions required for producing the rAAV of the disclosure
may be delivered to the packaging host cell using any appropriate
genetic element (vector). The selected genetic element may be
delivered by any suitable method, including those described herein.
The methods used to construct any embodiment of this disclosure are
known to those with skill in nucleic acid manipulation and include
genetic engineering, recombinant engineering, and synthetic
techniques. See, e.g., Sambrook et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. Similarly, methods of generating rAAV virions are well known
and the selection of a suitable method is not a limitation on the
present disclosure. See, e.g., K. Fisher et al, J. Virol.,
70:520-532 (1993) and U.S. Pat. No. 5,478,745.
In some embodiments, recombinant AAVs may be produced using the
triple transfection method (described in detail in U.S. Pat. No.
6,001,650). Typically, the recombinant AAVs are produced by
transfecting a host cell with an recombinant AAV vector (comprising
a transgene) to be packaged into AAV particles, an AAV helper
function vector, and an accessory function vector. An AAV helper
function vector encodes the "AAV helper function" sequences (i.e.,
rep and cap), which function in trans for productive AAV
replication and encapsidation. Preferably, the AAV helper function
vector supports efficient AAV vector production without generating
any detectable wild-type AAV virions (i.e., AAV virions containing
functional rep and cap genes). Non-limiting examples of vectors
suitable for use with the present disclosure include pHLP19,
described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector,
described in U.S. Pat. No. 6,156,303, the entirety of both
incorporated by reference herein. The accessory function vector
encodes nucleotide sequences for non-AAV derived viral and/or
cellular functions upon which AAV is dependent for replication
(i.e., "accessory functions"). The accessory functions include
those functions required for AAV replication, including, without
limitation, those moieties involved in activation of AAV gene
transcription, stage specific AAV mRNA splicing, AAV DNA
replication, synthesis of cap expression products, and AAV capsid
assembly. Viral-based accessory functions can be derived from any
of the known helper viruses such as adenovirus, herpesvirus (other
than herpes simplex virus type-1), and vaccinia virus.
In some aspects, the disclosure provides transfected host cells.
The term "transfection" is used to refer to the uptake of foreign
DNA by a cell, and a cell has been "transfected" when exogenous DNA
has been introduced inside the cell membrane. A number of
transfection techniques are generally known in the art. See, e.g.,
Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989)
Molecular Cloning, a laboratory manual, Cold Spring Harbor
Laboratories, New York, Davis et al. (1986) Basic Methods in
Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
Such techniques can be used to introduce one or more exogenous
nucleic acids, such as a nucleotide integration vector and other
nucleic acid molecules, into suitable host cells.
A "host cell" refers to any cell that harbors, or is capable of
harboring, a substance of interest. Often a host cell is a
mammalian cell. A host cell may be used as a recipient of an AAV
helper construct, an AAV minigene plasmid, an accessory function
vector, or other transfer DNA associated with the production of
recombinant AAVs. The term includes the progeny of the original
cell which has been transfected. Thus, a "host cell" as used herein
may refer to a cell which has been transfected with an exogenous
DNA sequence. It is understood that the progeny of a single
parental cell may not necessarily be completely identical in
morphology or in genomic or total DNA complement as the original
parent, due to natural, accidental, or deliberate mutation.
As used herein, the term "cell line" refers to a population of
cells capable of continuous or prolonged growth and division in
vitro. Often, cell lines are clonal populations derived from a
single progenitor cell. It is further known in the art that
spontaneous or induced changes can occur in karyotype during
storage or transfer of such clonal populations. Therefore, cells
derived from the cell line referred to may not be precisely
identical to the ancestral cells or cultures, and the cell line
referred to includes such variants.
As used herein, the terms "recombinant cell" refers to a cell into
which an exogenous DNA segment, such as DNA segment that leads to
the transcription of a biologically-active polypeptide or
production of a biologically active nucleic acid such as an RNA,
has been introduced.
As used herein, the term "vector" includes any genetic element,
such as a plasmid, phage, transposon, cosmid, chromosome,
artificial chromosome, virus, virion, etc., which is capable of
replication when associated with the proper control elements and
which can transfer gene sequences between cells. Thus, the term
includes cloning and expression vehicles, as well as viral vectors.
In some embodiments, useful vectors are contemplated to be those
vectors in which the nucleic acid segment to be transcribed is
positioned under the transcriptional control of a promoter. A
"promoter" refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required
to initiate the specific transcription of a gene. The phrases
"operatively positioned," "under control" or "under transcriptional
control" means that the promoter is in the correct location and
orientation in relation to the nucleic acid to control RNA
polymerase initiation and expression of the gene. The term
"expression vector or construct" means any type of genetic
construct containing a nucleic acid in which part or all of the
nucleic acid encoding sequence is capable of being transcribed. In
some embodiments, expression includes transcription of the nucleic
acid, for example, to generate a biologically-active polypeptide
product or functional RNA (e.g., guide RNA) from a transcribed
gene.
The foregoing methods for packaging recombinant vectors in desired
AAV capsids to produce the rAAVs of the disclosure are not meant to
be limiting and other suitable methods will be apparent to the
skilled artisan.
Recombinant AAV Vectors
"Recombinant AAV (rAAV) vectors" of the disclosure are typically
composed of, at a minimum, a transgene and its regulatory
sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It
is this recombinant AAV vector which is packaged into a capsid
protein and delivered to a selected target cell. In some
embodiments, the transgene is a nucleic acid sequence, heterologous
to the vector sequences, which encodes a polypeptide, protein,
functional RNA molecule (e.g., gRNA) or other gene product, of
interest. The nucleic acid coding sequence is operatively linked to
regulatory components in a manner which permits transgene
transcription, translation, and/or expression in a cell of a target
tissue.
In some embodiments, the instant disclosure relates to a
recombinant AAV (rAAV) comprising a capsid protein having an
N-terminally grafted nuclease, wherein the N-terminally grafted
nuclease is present only in the VP2 capsid protein. In some
embodiments, the rAAV comprises a capsid protein having an amino
acid sequence represented by SEQ ID NO: 4.
The AAV sequences of the vector typically comprise the cis-acting
5' and 3' inverted terminal repeat sequences (See, e.g., B. J.
Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press,
pp. 155 168 (1990)). The ITR sequences are about 145 bp in length.
Preferably, substantially the entire sequences encoding the ITRs
are used in the molecule, although some degree of minor
modification of these sequences is permissible. The ability to
modify these ITR sequences is within the skill of the art. (See,
e.g., texts such as Sambrook et al, "Molecular Cloning. A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York
(1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An
example of such a molecule employed in the present disclosure is a
"cis-acting" plasmid containing the transgene, in which the
selected transgene sequence and associated regulatory elements are
flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences
may be obtained from any known AAV, including presently identified
mammalian AAV types.
In addition to the major elements identified above for the
recombinant AAV vector, the vector also includes conventional
control elements necessary which are operably linked to the
transgene in a manner which permits its transcription, translation
and/or expression in a cell transfected with the plasmid vector or
infected with the virus produced by the disclosure. As used herein,
"operably linked" sequences include both expression control
sequences that are contiguous with the gene of interest and
expression control sequences that act in trans or at a distance to
control the gene of interest.
Expression control sequences include appropriate transcription
initiation, termination, promoter and enhancer sequences; efficient
RNA processing signals such as splicing and polyadenylation (polyA)
signals; sequences that stabilize cytoplasmic mRNA; sequences that
enhance translation efficiency (i.e., Kozak consensus sequence);
sequences that enhance protein stability; and when desired,
sequences that enhance secretion of the encoded product. A great
number of expression control sequences, including promoters which
are native, constitutive, inducible and/or tissue-specific, are
known in the art and may be utilized.
As used herein, a nucleic acid sequence (e.g., coding sequence) and
regulatory sequences are said to be "operably" linked when they are
covalently linked in such a way as to place the expression or
transcription of the nucleic acid sequence under the influence or
control of the regulatory sequences. If it is desired that the
nucleic acid sequences be translated into a functional protein, two
DNA sequences are said to be operably linked if induction of a
promoter in the 5' regulatory sequences results in the
transcription of the coding sequence and if the nature of the
linkage between the two DNA sequences does not (1) result in the
introduction of a frame-shift mutation, (2) interfere with the
ability of the promoter region to direct the transcription of the
coding sequences, or (3) interfere with the ability of the
corresponding RNA transcript to be translated into a protein. Thus,
a promoter region would be operably linked to a nucleic acid
sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide. Similarly two or more coding regions are operably
linked when they are linked in such a way that their transcription
from a common promoter results in the expression of two or more
proteins having been translated in frame. In some embodiments,
operably linked coding sequences yield a fusion protein. In some
embodiments, operably linked coding sequences yield a functional
RNA (e.g., gRNA).
For nucleic acids encoding proteins, a polyadenylation sequence
generally is inserted following the transgene sequences and before
the 3' AAV ITR sequence. A rAAV construct useful in the present
disclosure may also contain an intron, desirably located between
the promoter/enhancer sequence and the transgene. One possible
intron sequence is derived from SV-40, and is referred to as the
SV-40 T intron sequence. Another vector element that may be used is
an internal ribosome entry site (IRES). An IRES sequence is used to
produce more than one polypeptide from a single gene transcript. An
IRES sequence would be used to produce a protein that contain more
than one polypeptide chains. Selection of these and other common
vector elements are conventional and many such sequences are
available [see, e.g., Sambrook et al, and references cited therein
at, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et
al., Current Protocols in Molecular Biology, John Wiley & Sons,
New York, 1989]. In some embodiments, a Foot and Mouth Disease
Virus 2A sequence is included in polyprotein; this is a small
peptide (approximately 18 amino acids in length) that has been
shown to mediate the cleavage of polyproteins (Ryan, M D et al.,
EMBO, 1994; 4: 928-933; Mattion, N M et al., J Virology, November
1996; p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8:
864-873; and Halpin, C et al., The Plant Journal, 1999; 4:
453-459). The cleavage activity of the 2A sequence has previously
been demonstrated in artificial systems including plasmids and gene
therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO,
1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996;
p. 8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and
Halpin, C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P
et al., Gene Therapy, 1999; 6: 198-208; de Felipe, P et al., Human
Gene Therapy, 2000; 11: 1921-1931; and Klump, H et al., Gene
Therapy, 2001; 8: 811-817).
The precise nature of the regulatory sequences needed for gene
expression in host cells may vary between species, tissues or cell
types, but shall in general include, as necessary, 5'
non-transcribed and 5' non-translated sequences involved with the
initiation of transcription and translation respectively, such as a
TATA box, capping sequence, CAAT sequence, enhancer elements, and
the like. Especially, such 5' non-transcribed regulatory sequences
will include a promoter region that includes a promoter sequence
for transcriptional control of the operably joined gene. Regulatory
sequences may also include enhancer sequences or upstream activator
sequences as desired. The vectors of the disclosure may optionally
include 5' leader or signal sequences. The choice and design of an
appropriate vector is within the ability and discretion of one of
ordinary skill in the art.
Examples of constitutive promoters include, without limitation, the
retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with
the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally
with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530
(1985)], the SV40 promoter, the dihydrofolate reductase promoter,
the .beta.-actin promoter, the phosphoglycerol kinase (PGK)
promoter, and the EF1.alpha. promoter [Invitrogen].
Inducible promoters allow regulation of gene expression and can be
regulated by exogenously supplied compounds, environmental factors
such as temperature, or the presence of a specific physiological
state, e.g., acute phase, a particular differentiation state of the
cell, or in replicating cells only. Inducible promoters and
inducible systems are available from a variety of commercial
sources, including, without limitation, Invitrogen, Clontech and
Ariad. Many other systems have been described and can be readily
selected by one of skill in the art. Examples of inducible
promoters regulated by exogenously supplied promoters include the
zinc-inducible sheep metallothionine (MT) promoter, the
dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV)
promoter, the T7 polymerase promoter system (WO 98/10088); the
ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA,
93:3346-3351 (1996)), the tetracycline-repressible system (Gossen
et al, Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the
tetracycline-inducible system (Gossen et al, Science, 268:1766-1769
(1995), see also Harvey et al, Curr. Opin. Chem. Biol., 2:512-518
(1998)), the RU486-inducible system (Wang et al, Nat. Biotech.,
15:239-243 (1997) and Wang et al, Gene Ther., 4:432-441 (1997)) and
the rapamycin-inducible system (Magari et al, J. Clin. Invest.,
100:2865-2872 (1997)). Still other types of inducible promoters
which may be useful in this context are those which are regulated
by a specific physiological state, e.g., temperature, acute phase,
a particular differentiation state of the cell, or in replicating
cells only.
In another embodiment, the native promoter for the transgene will
be used. The native promoter may be preferred when it is desired
that expression of the transgene should mimic the native
expression. The native promoter may be used when expression of the
transgene must be regulated temporally or developmentally, or in a
tissue-specific manner, or in response to specific transcriptional
stimuli. In a further embodiment, other native expression control
elements, such as enhancer elements, polyadenylation sites or Kozak
consensus sequences may also be used to mimic the native
expression.
In some embodiments, the regulatory sequences impart
tissue-specific gene expression capabilities. In some cases, the
tissue-specific regulatory sequences bind tissue-specific
transcription factors that induce transcription in a tissue
specific manner. Such tissue-specific regulatory sequences (e.g.,
promoters, enhancers, etc.) are well known in the art. Exemplary
tissue-specific regulatory sequences include, but are not limited
to the following tissue specific promoters: a liver-specific
thyroxin binding globulin (TBG) promoter, an insulin promoter, a
glucagon promoter, a somatostatin promoter, a pancreatic
polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine
kinase (MCK) promoter, a mammalian desmin (DES) promoter, a
.alpha.-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin
T (cTnT) promoter. Other exemplary promoters include Beta-actin
promoter, hepatitis B virus core promoter, Sandig et al., Gene
Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot
et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin
promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone
sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64
(1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8
(1998); immunoglobulin heavy chain promoter; T cell receptor
.alpha.-chain promoter, neuronal such as neuron-specific enolase
(NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15
(1993)), neurofilament light-chain gene promoter (Piccioli et al.,
Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the
neuron-specific vgf gene promoter (Piccioli et al., Neuron,
15:373-84 (1995)), among others which will be apparent to the
skilled artisan.
In some embodiments, one or more bindings sites for one or more of
miRNAs are incorporated in a transgene of a rAAV vector, to inhibit
the expression of the transgene in one or more tissues of an
subject harboring the transgene. The skilled artisan will
appreciate that binding sites may be selected to control the
expression of a transgene in a tissue specific manner. For example,
binding sites for the liver-specific miR-122 may be incorporated
into a transgene to inhibit expression of that transgene in the
liver. The target sites in the mRNA may be in the 5' UTR, the 3'
UTR or in the coding region. Typically, the target site is in the
3' UTR of the mRNA. Furthermore, the transgene may be designed such
that multiple miRNAs regulate the mRNA by recognizing the same or
multiple sites. The presence of multiple miRNA binding sites may
result in the cooperative action of multiple RISCs and provide
highly efficient inhibition of expression. The target site sequence
may comprise a total of 5-100, 10-60, or more nucleotides. The
target site sequence may comprise at least 5 nucleotides of the
sequence of a target gene binding site.
Recombinant AAV Vector: Transgene Coding Sequences
The composition of the transgene sequence of the rAAV vector will
depend upon the use to which the resulting vector will be put. For
example, one type of transgene sequence includes a reporter
sequence, which upon expression produces a detectable signal. In
another example, the transgene encodes a therapeutic protein or
therapeutic functional RNA. In another example, the transgene
encodes a protein or functional RNA that is intended to be used for
research purposes, e.g., to create a somatic transgenic animal
model harboring the transgene, e.g., to study the function of the
transgene product. In another example, the transgene encodes a
protein or functional RNA that is intended to be used to create an
animal model of disease. Appropriate transgene coding sequences
will be apparent to the skilled artisan.
Also contemplated herein are methods of delivering a transgene to a
subject using the rAAVs described herein. In some embodiments, the
instant disclosure relates to a method for delivering a transgene
to a subject comprising administering a rAAV to a subject, wherein
the rAAV comprises: (i) a capsid protein having a terminally
grafted nuclease, e.g., a nuclease having a sequence set forth as
SEQ ID NO: 2, and optionally (ii) at least one transgene, e.g., a
transgene encoding a gRNA, and wherein the rAAV infects cells of a
target tissue of the subject. In some embodiments of the method, at
least one transgene encodes a single guide RNA, a CRISPR RNA
(crRNA), and/or a trans-activating crRNA (tracrRNA).
In some embodiments, the rAAV vectors may comprise a transgene,
wherein the transgene is a gRNA. In some embodiments, the gRNA
targets a nucleic acid sequence that causes disease in a subject.
For example, expression of the huntingtin (Htt) gene causes
Huntington's disease. Without wishing to be bound by any particular
theory, a gRNA targeting the Htt gene directs Cas9 cleavage of the
gene, thereby preventing its expression. Other similar genes
(disease-associated or otherwise) can be targeted.
Recombinant AAV Administration Methods
The rAAVs may be delivered to a subject in compositions according
to any appropriate methods known in the art. The rAAV, preferably
suspended in a physiologically compatible carrier (i.e., in a
composition), may be administered to a subject, i.e. host animal,
such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow,
goat, pig, guinea pig, hamster, chicken, turkey, or a non-human
primate (e.g., Macaque). In some embodiments a host animal does not
include a human.
Delivery of the rAAVs to a mammalian subject may be by, for
example, intramuscular injection or by administration into the
bloodstream of the mammalian subject. Administration into the
bloodstream may be by injection into a vein, an artery, or any
other vascular conduit. In some embodiments, the rAAVs are
administered into the bloodstream by way of isolated limb
perfusion, a technique well known in the surgical arts, the method
essentially enabling the artisan to isolate a limb from the
systemic circulation prior to administration of the rAAV virions. A
variant of the isolated limb perfusion technique, described in U.S.
Pat. No. 6,177,403, can also be employed by the skilled artisan to
administer the virions into the vasculature of an isolated limb to
potentially enhance transduction into muscle cells or tissue.
Moreover, in certain instances, it may be desirable to deliver the
virions to the CNS of a subject. By "CNS" is meant all cells and
tissue of the brain and spinal cord of a vertebrate. Thus, the term
includes, but is not limited to, neuronal cells, glial cells,
astrocytes, cereobrospinal fluid (CSF), interstitial spaces, bone,
cartilage and the like. Recombinant AAVs may be delivered directly
to the CNS or brain by injection into, e.g., the ventricular
region, as well as to the striatum (e.g., the caudate nucleus or
putamen of the striatum), spinal cord and neuromuscular junction,
or cerebellar lobule, with a needle, catheter or related device,
using neurosurgical techniques known in the art, such as by
stereotactic injection (see, e.g., Stein et al., J Virol
73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000;
Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and
Davidson, Hum. Gene Ther. 11:2315-2329, 2000).
Aspects of the instant disclosure relate to compositions comprising
a recombinant AAV comprising a capsid protein having a terminally
grafted (e.g., N-terminally grafted or C-terminally grafted)
nuclease. In some embodiments, the nuclease is terminally grafted
onto a capsid protein. In some embodiments, the a terminally
grafted nuclease is present on all three capsid proteins (e.g. VP1,
VP2, VP3) of the rAAV. In some embodiments, the terminally grafted
nuclease is present on two of the capsid proteins (e.g. VP2 and
VP3) of the rAAV. In some embodiments, the terminally grafted
nuclease is present on a single capsid protein of the rAAV. In some
embodiments, the terminally grafted nuclease is present on the VP2
capsid protein of the rAAV. In some embodiments, the composition
further comprises a pharmaceutically acceptable carrier.
The compositions of the disclosure may comprise an rAAV alone, or
in combination with one or more other viruses (e.g., a second rAAV
encoding having one or more different transgenes). In some
embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more different rAAVs each having one or more different
transgenes.
Suitable carriers may be readily selected by one of skill in the
art in view of the indication for which the rAAV is directed. For
example, one suitable carrier includes saline, which may be
formulated with a variety of buffering solutions (e.g., phosphate
buffered saline). Other exemplary carriers include sterile saline,
lactose, sucrose, calcium phosphate, gelatin, dextran, agar,
pectin, peanut oil, sesame oil, and water. The selection of the
carrier is not a limitation of the present disclosure.
Optionally, the compositions of the disclosure may contain, in
addition to the rAAV and carrier(s), other conventional
pharmaceutical ingredients, such as preservatives, or chemical
stabilizers. Suitable exemplary preservatives include
chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide,
propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and
parachlorophenol. Suitable chemical stabilizers include gelatin and
albumin.
The rAAVS are administered in sufficient amounts to transfect the
cells of a desired tissue and to provide sufficient levels of gene
transfer and expression without undue adverse effects. Conventional
and pharmaceutically acceptable routes of administration include,
but are not limited to, direct delivery to the selected organ
(e.g., intraportal delivery to the liver), oral, inhalation
(including intranasal and intratracheal delivery), intraocular,
intravenous, intramuscular, subcutaneous, intradermal,
intratumoral, and other parental routes of administration. Routes
of administration may be combined, if desired.
The dose of rAAV virions required to achieve a particular
"therapeutic effect," e.g., the units of dose in genome copies/per
kilogram of body weight (GC/kg), will vary based on several factors
including, but not limited to: the route of rAAV virion
administration, the level of gene or RNA expression required to
achieve a therapeutic effect, the specific disease or disorder
being treated, and the stability of the gene or RNA product. One of
skill in the art can readily determine a rAAV virion dose range to
treat a patient having a particular disease or disorder based on
the aforementioned factors, as well as other factors that are well
known in the art.
An effective amount of an rAAV is an amount sufficient to target
infect an animal, target a desired tissue. In some embodiments, an
effective amount of an rAAV is an amount sufficient to produce a
stable somatic transgenic animal model. The effective amount will
depend primarily on factors such as the species, age, weight,
health of the subject, and the tissue to be targeted, and may thus
vary among animal and tissue. For example, an effective amount of
the rAAV is generally in the range of from about 1 ml to about 100
ml of solution containing from about 10.sup.9 to 10.sup.16 genome
copies. In some cases, a dosage between about 10.sup.11 to
10.sup.13 rAAV genome copies is appropriate. In certain
embodiments, 10.sup.12 or 10.sup.13 rAAV genome copies is effective
to target heart, liver, and pancreas tissues. In some cases, stable
transgenic animals are produced by multiple doses of an rAAV.
In some embodiments, rAAV compositions are formulated to reduce
aggregation of AAV particles in the composition, particularly where
high rAAV concentrations are present (e.g., .about.10.sup.13 GC/ml
or more). Methods for reducing aggregation of rAAVs are well known
in the art and, include, for example, addition of surfactants, pH
adjustment, salt concentration adjustment, etc. (See, e.g., Wright
F R, et al., Molecular Therapy (2005) 12, 171-178, the contents of
which are incorporated herein by reference.)
Formulation of pharmaceutically-acceptable excipients and carrier
solutions is well-known to those of skill in the art, as is the
development of suitable dosing and treatment regimens for using the
particular compositions described herein in a variety of treatment
regimens.
Typically, these formulations may contain at least about 0.1% of
the active compound or more, although the percentage of the active
ingredient(s) may, of course, be varied and may conveniently be
between about 1 or 2% and about 70% or 80% or more of the weight or
volume of the total formulation. Naturally, the amount of active
compound in each therapeutically-useful composition may be prepared
is such a way that a suitable dosage will be obtained in any given
unit dose of the compound. Factors such as solubility,
bioavailability, biological half-life, route of administration,
product shelf life, as well as other pharmacological considerations
will be contemplated by one skilled in the art of preparing such
pharmaceutical formulations, and as such, a variety of dosages and
treatment regimens may be desirable.
In certain circumstances it will be desirable to deliver the
rAAV-based therapeutic constructs in suitably formulated
pharmaceutical compositions disclosed herein either subcutaneously,
intraopancreatically, intranasally, parenterally, intravenously,
intramuscularly, intrathecally, or orally, intraperitoneally, or by
inhalation. In some embodiments, the administration modalities as
described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363
(each specifically incorporated herein by reference in its
entirety) may be used to deliver rAAVs. In some embodiments, a
preferred mode of administration is by portal vein injection.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. Dispersions may also be prepared in glycerol, liquid
polyethylene glycols, and mixtures thereof and in oils. Under
ordinary conditions of storage and use, these preparations contain
a preservative to prevent the growth of microorganisms. In many
cases the form is sterile and fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (e.g., glycerol, propylene glycol,
and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and/or vegetable oils. Proper fluidity may be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
For administration of an injectable aqueous solution, for example,
the solution may be suitably buffered, if necessary, and the liquid
diluent first rendered isotonic with sufficient saline or glucose.
These particular aqueous solutions are especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal
administration. In this connection, a sterile aqueous medium that
can be employed will be known to those of skill in the art. For
example, one dosage may be dissolved in 1 ml of isotonic NaCl
solution and either added to 1000 ml of hypodermoclysis fluid or
injected at the proposed site of infusion, (see for example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038
and 1570-1580). Some variation in dosage will necessarily occur
depending on the condition of the host. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual host.
Sterile injectable solutions are prepared by incorporating the
active rAAV in the required amount in the appropriate solvent with
various of the other ingredients enumerated herein, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the various sterilized active ingredients
into a sterile vehicle which contains the basic dispersion medium
and the required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum-drying and freeze-drying techniques which yield a powder of
the active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
The rAAV compositions disclosed herein may also be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug-release capsules,
and the like.
As used herein, "carrier" includes any and all solvents, dispersion
media, vehicles, coatings, diluents, antibacterial and antifungal
agents, isotonic and absorption delaying agents, buffers, carrier
solutions, suspensions, colloids, and the like. The use of such
media and agents for pharmaceutical active substances is well known
in the art. Supplementary active ingredients can also be
incorporated into the compositions. The phrase
"pharmaceutically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a host.
Delivery vehicles such as liposomes, nanocapsules, microparticles,
microspheres, lipid particles, vesicles, and the like, may be used
for the introduction of the compositions of the present disclosure
into suitable host cells. In particular, the rAAV vector delivered
transgenes may be formulated for delivery either encapsulated in a
lipid particle, a liposome, a vesicle, a nanosphere, or a
nanoparticle or the like.
Such formulations may be preferred for the introduction of
pharmaceutically acceptable formulations of the nucleic acids or
the rAAV constructs disclosed herein. The formation and use of
liposomes is generally known to those of skill in the art.
Recently, liposomes were developed with improved serum stability
and circulation half-times (U.S. Pat. No. 5,741,516). Further,
various methods of liposome and liposome like preparations as
potential drug carriers have been described (U.S. Pat. Nos.
5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).
Liposomes have been used successfully with a number of cell types
that are normally resistant to transfection by other procedures. In
addition, liposomes are free of the DNA length constraints that are
typical of viral-based delivery systems. Liposomes have been used
effectively to introduce genes, drugs, radiotherapeutic agents,
viruses, transcription factors and allosteric effectors into a
variety of cultured cell lines and animals. In addition, several
successful clinical trials examining the effectiveness of
liposome-mediated drug delivery have been completed.
Liposomes are formed from phospholipids that are dispersed in an
aqueous medium and spontaneously form multilamellar concentric
bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs
generally have diameters of from 25 nm to 4 .mu.m. Sonication of
MLVs results in the formation of small unilamellar vesicles (SUVs)
with diameters in the range of 200 to 500 ANG., containing an
aqueous solution in the core.
Alternatively, nanocapsule formulations of the rAAV may be used.
Nanocapsules can generally entrap substances in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use.
In addition to the methods of delivery described above, the
following techniques are also contemplated as alternative methods
of delivering the rAAV compositions to a host. Sonophoresis (e.g.,
ultrasound) has been used and described in U.S. Pat. No. 5,656,016
as a device for enhancing the rate and efficacy of drug permeation
into and through the circulatory system. Other drug delivery
alternatives contemplated are intraosseous injection (U.S. Pat. No.
5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic
formulations (Bourlais et al., 1998), transdermal matrices (U.S.
Pat. Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery
(U.S. Pat. No. 5,697,899).
Kits and Related Compositions
The agents described herein may, in some embodiments, be assembled
into pharmaceutical or diagnostic or research kits to facilitate
their use in therapeutic, diagnostic or research applications. A
kit may include one or more containers housing the components of
the disclosure and instructions for use. Specifically, such kits
may include one or more agents described herein, along with
instructions describing the intended application and the proper use
of these agents. In certain embodiments agents in a kit may be in a
pharmaceutical formulation and dosage suitable for a particular
application and for a method of administration of the agents. Kits
for research purposes may contain the components in appropriate
concentrations or quantities for running various experiments.
In some embodiments, the instant disclosure relates to a kit for
producing a rAAV, the kit comprising a container housing an
isolated nucleic acid having a sequence of SEQ ID NO: 1 or SEQ ID
NO: 3. In some embodiments, the kit further comprises instructions
for producing the rAAV. In some embodiments, the kit further
comprises at least one container housing a recombinant AAV vector,
wherein the recombinant AAV vector comprises a transgene.
In some embodiments, the instant disclosure relates to a kit
comprising a container housing a recombinant AAV having an isolated
AAV capsid protein having an amino acid sequence as set forth in
any of SEQ ID NO: 4.
The kit may be designed to facilitate use of the methods described
herein by researchers and can take many forms. Each of the
compositions of the kit, where applicable, may be provided in
liquid form (e.g., in solution), or in solid form, (e.g., a dry
powder). In certain cases, some of the compositions may be
constitutable or otherwise processable (e.g., to an active form),
for example, by the addition of a suitable solvent or other species
(for example, water or a cell culture medium), which may or may not
be provided with the kit. As used herein, "instructions" can define
a component of instruction and/or promotion, and typically involve
written instructions on or associated with packaging of the
disclosure. Instructions also can include any oral or electronic
instructions provided in any manner such that a user will clearly
recognize that the instructions are to be associated with the kit,
for example, audiovisual (e.g., videotape, DVD, etc.), Internet,
and/or web-based communications, etc. The written instructions may
be in a form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which instructions can also reflects approval by the agency of
manufacture, use or sale for animal administration.
The kit may contain any one or more of the components described
herein in one or more containers. As an example, in one embodiment,
the kit may include instructions for mixing one or more components
of the kit and/or isolating and mixing a sample and applying to a
subject. The kit may include a container housing agents described
herein. The agents may be in the form of a liquid, gel or solid
(powder). The agents may be prepared sterilely, packaged in syringe
and shipped refrigerated. Alternatively it may be housed in a vial
or other container for storage. A second container may have other
agents prepared sterilely. Alternatively the kit may include the
active agents premixed and shipped in a syringe, vial, tube, or
other container. The kit may have one or more or all of the
components required to administer the agents to an animal, such as
a syringe, topical application devices, or iv needle tubing and
bag, particularly in the case of the kits for producing specific
somatic animal models.
In some cases, the methods involve transfecting cells with total
cellular DNAs isolated from the tissues that potentially harbor
proviral AAV genomes at very low abundance and supplementing with
helper virus function (e.g., adenovirus) to trigger and/or boost
AAV rep and cap gene transcription in the transfected cell. In some
cases, RNA from the transfected cells provides a template for
RT-PCR amplification of cDNA and the detection of novel AAVs. In
cases where cells are transfected with total cellular DNAs isolated
from the tissues that potentially harbor proviral AAV genomes, it
is often desirable to supplement the cells with factors that
promote AAV gene transcription. For example, the cells may also be
infected with a helper virus, such as an Adenovirus or a Herpes
Virus. In a specific embodiment, the helper functions are provided
by an adenovirus. The adenovirus may be a wild-type adenovirus, and
may be of human or non-human origin, preferably non-human primate
(NHP) origin. Similarly adenoviruses known to infect non-human
animals (e.g., chimpanzees, mouse) may also be employed in the
methods of the disclosure (See, e.g., U.S. Pat. No. 6,083,716). In
addition to wild-type adenoviruses, recombinant viruses or
non-viral vectors (e.g., plasmids, episomes, etc.) carrying the
necessary helper functions may be utilized. Such recombinant
viruses are known in the art and may be prepared according to
published techniques. See, e.g., U.S. Pat. Nos. 5,871,982 and
6,251,677, which describe a hybrid Ad/AAV virus. A variety of
adenovirus strains are available from the American Type Culture
Collection, Manassas, Va., or available by request from a variety
of commercial and institutional sources. Further, the sequences of
many such strains are available from a variety of databases
including, e.g., PubMed and GenBank.
Cells may also be transfected with a vector (e.g., helper vector)
which provides helper functions to the AAV. The vector providing
helper functions may provide adenovirus functions, including, e.g.,
E1a, E1b, E2a, E4ORF6. The sequences of adenovirus gene providing
these functions may be obtained from any known adenovirus serotype,
such as serotypes 2, 3, 4, 7, 12 and 40, and further including any
of the presently identified human types known in the art. Thus, in
some embodiments, the methods involve transfecting the cell with a
vector expressing one or more genes necessary for AAV replication,
AAV gene transcription, and/or AAV packaging.
In some cases, a capsid gene can be used to construct and package
recombinant AAV vectors, using methods well known in the art, to
determine functional characteristics associated with the novel
capsid protein encoded by the gene. For example, novel isolated
capsid genes can be used to construct and package recombinant AAV
(rAAV) vectors comprising a reporter gene (e.g., B-Galactosidase,
GFP, Luciferase, etc.). The rAAV vector can then be delivered to an
animal (e.g., mouse) and the tissue targeting properties of the
novel isolated capsid gene can be determined by examining the
expression of the reporter gene in various tissues (e.g., heart,
liver, kidneys) of the animal. Other methods for characterizing the
novel isolated capsid genes are disclosed herein and still others
are well known in the art.
The kit may have a variety of forms, such as a blister pouch, a
shrink wrapped pouch, a vacuum sealable pouch, a sealable
thermoformed tray, or a similar pouch or tray form, with the
accessories loosely packed within the pouch, one or more tubes,
containers, a box or a bag. The kit may be sterilized after the
accessories are added, thereby allowing the individual accessories
in the container to be otherwise unwrapped. The kits can be
sterilized using any appropriate sterilization techniques, such as
radiation sterilization, heat sterilization, or other sterilization
methods known in the art. The kit may also include other
components, depending on the specific application, for example,
containers, cell media, salts, buffers, reagents, syringes,
needles, a fabric, such as gauze, for applying or removing a
disinfecting agent, disposable gloves, a support for the agents
prior to administration etc.
The instructions included within the kit may involve methods for
detecting a latent AAV in a cell. In addition, kits of the
disclosure may include, instructions, a negative and/or positive
control, containers, diluents and buffers for the sample, sample
preparation tubes and a printed or electronic table of reference
AAV sequence for sequence comparisons.
EXAMPLES
Overview
To avoid the potential genotoxicity due to prolonged expression
gene editing components, an endonuclease protein is transiently
delivered and degrades naturally in the cell. Specifically, AAV
capsid is used as a delivery vehicle to carry a Cas9 protein or
other designer nuclease protein. AAV capsid consists of 60 copies
of three capsid proteins, VP1, VP2 and VP3, at a ratio of 1:1:18.
Although AAV capsid adopts a tightly packed structure, it has been
shown that the VP2 protein with an N-terminal fusion protein can be
incorporated into AAV capsid, and such a chimeric AAV is
infectious.
Example 1
Results provided herein indicate that when Cas9 is fused to the
N-terminus of VP2 (FIG. 1A), the resulting Cas9-VP2 fusion protein
is functional.
A plasmid expressing S. pyogenes Cas9 (SpCas9; SEQ ID NOs: 1 and 2)
fused with AAV2 VP2 was constructed (FIG. 1A). The resulting
construct is represented by SEQ ID NO: 3 and the fusion protein is
represented by SEQ ID NO: 4. Transfection of the construct into
HEK293 cells yields a fusion protein product of expected size, as
demonstrated by western blotting (FIG. 1B). A fluorescence reporter
assay as illustrated in FIG. 2A was used to test if SpCas9-VP2 can
function in gene editing. In the reporter construct, the GFP is
disrupted by an insertion. The downstream out-of-frame T2A, when
shifted to in-frame, mediates translation termination and
re-initiation to produce mCherry reporter protein. In the presence
of the sgRNA targeting the insertion in the GFP sequence and a
functional SpCas9, indels by NHEJ shift the downstream T2A and
mCherry to in-frame, thus giving mCherry fluorescence signal. Using
this reporter system, SpCas9-VP2 induction of NHEJ by
co-transfection in HEK293 cells was tested (FIG. 2B). Negative
control cells expressing SpCas9 only or sgRNA only did not induce
mCherry signal. Positive control cells, co-expressing sgRNA and
SpCas9 yielded mCherry signal. When sgRNA and the SpCas9-VP2 fusion
were co-expressed, mCherry fluorescence was also observed,
demonstrating that the SpCas-VP2 fusion protein behaves similarly
as SpCas9 in inducing gene editing and NHEJ (FIG. 2B).
SpCas-VP2 can be also incorporated into AAV2 capsid to form AAV
virion. The start codon of VP2 is mutated in the cap gene from the
trans AAV production plasmid. The omission of VP2 expression from
this plasmid in HEK293 cells is validated by western blotting using
an antibody targeting a C-terminal epitope shared by VP1, VP2 and
VP3. Small-scale AAV production is performed using the VP2
null-trans plasmid and the SpCas9-VP2 in replacement of the
original trans plasmid to examine the presence of Cas9 protein
covalently linked to the outer surface of AAV2 virion. ELISA is
performed using antibodies recognizing a fully assembled AAV2
virion and the HA-tagged SpCas9. Alternatively, immuno electron
microscopy is performed to visualize the presence of HA-tagged
SpCas9 immunoreactivity outside of AAV2 virion. Next, a small-scale
AAV production-infection assay is performed to validate that
SpCas9-AAV delivers the SpCas9 into HEK293 cells and mediates gene
editing. The same reporter system as illustrated in FIG. 2B is used
for this assay.
Serials of in vivo experiments using SpCas9-AAV2 expressing EGFP
and sgRNA targeting the mouse ROSA26 locus
(SpCas9-AAV2-EGFP-sgROSA26) obtained from large-scale production
are next performed. The tropism of SpCas9-AAV2 is characterized in
mice by systemic delivery. Wild-type C57BL/6J mice are injected
with SpCas9-AAV2-EGFP-sgROSA26 at postnatal day 1 (P1) via facial
vein and at 8 weeks old via tail vein, respectively. The mice are
sacrificed 3 weeks after injection and fixed. Tissues including
liver, heart, skeletal muscle, pancreas, adrenal gland, kidneys,
spleen, brain, and spinal cord are analyzed for EGFP expression by
immunofluorescence staining. The best transduced tissue(s) are
selected to demonstrate SpCas9-AAV mediated gene editing of ROSA26
locus in vivo in another group of mice treated in the same manner,
from which fresh tissues are harvested and genomic DNA extracted.
The gene editing events represented by random indels near the sgRNA
targeting site in the ROSA26 locus are investigated using Surveyor
assay and single DNA molecule sequencing.
To demonstrate the improved safety profile of the SpCas9
transiently delivered using SpCas9-AAV2 and contrast with prolonged
expression of SpCas9 from a conventional rAAV2 vector, SpCas9-AAV2
are packaged with transgene cassettes expressing sgRNAs with
reported off-target effects in mouse genome, and inject into mice.
The gene editing events at both on- and off-target genomic DNA loci
are analyzed by Surveyor assay and single DNA molecule sequencing.
Transient delivery of SpCas9 significantly reduces the chance of
off-target effect.
Example 2
Intein-mediated protein trans-splicing (PTS) is used to fuse SpCas9
protein with VP2 after AAV assembly as illustrated in FIG. 3A. For
example, the naturally split intein Npu DnaE, which has the most
robust trans-splicing activity identified so far, is used to fuse
SpCas9 protein with VP2 by PTS. The SpCas9 carries IntN, and VP2
carries IntC. Since IntC comprises only 36 amino acid residues, an
IntC-VP2 fusion is amenable to AAV assembly. First, IntC-AAV2
virion and SpCas9-IntN protein are produced and purified
separately, and then incubated to allow for PTS to occur in vitro.
Intein-mediated PTS is a spontaneous reaction and does not require
other co-factors. The fast kinetic nature of Npu DnaE split intein
produces SpCas9-AAV2 fusion protein rapidly. The fusion protein is
further purified by dialysis.
Alternatively, IntC is fused with a truncated C-terminal portion of
SpCas9 onto AAV2 capsid to allow for in vivo transduction. The rest
portion of SpCas9 and IntN are encoded by a transgene expression
cassette as rAAV genome (FIG. 3B). Co-delivery of the two portions
of SpCas9 reconstitutes the full-length, functional SpCas9.
Importantly, as the IntC-SpCas9C protein is degraded naturally, the
long-term expression of SpCas9N-IntN only is non-functional, thus
mitigating off-target effects.
FIG. 4 shows one example of an expression construct comprising a
nucleic acid sequence encoding SpCas9 nuclease N-terminally fused
to VP2 capsid protein.
Example 3
Gene editing platforms, such as the Cas9/sgRNA system, have been
shown to induce off-target DNA double-stranded breaks (DSBs)
throughout genomes, which is associated with toxicity. Such
off-target effects not only stem from ambiguity of DNA sequence
recognition by nucleases, but also can be attributed to the
prolonged presence of an active gene editing system in a given
cell. As a result, off-target DSBs accumulate over time, and
ultimately lead to genotoxicity. To mitigate the potential toxicity
due to prolonged expression of gene editing system in vivo,
transient delivery of endonuclease protein, which induces permanent
gene editing followed by natural degradation of the endonuclease,
was examined. Specifically, the VP2 protein of AAV capsid was used
as a protein delivery vehicle to ferry the Cas9 protein in
vivo.
A sensitive gene editing reporter plasmid was constructed.
Co-transfection of the reporter plasmid and a plasmid expressing
the SpCas9-VP2 fusion protein induced gene editing in HEK293 cells.
An rAAV packaging system was modified to include a plasmid
expressing VP1 and VP3, and another plasmid expressing either the
SpCas9-VP2 fusion protein or the EGFP-VP2 fusion protein. EGFP-AAV2
(EGFP protein grafted on the AAV2 capsid) was successfully
produced. However, rAAV particles carrying SpCas9 protein were not
produced, likely because the large size of SpCas9 protein
interfered with the AAV packaging process.
The transgene encoding SpCas9 was split into halves to utilize
split intein-mediated protein trans-splicing (PTS) to transiently
reconstitute the full-length SpCas9 (FIG. 3A). When the two parts
of a split intein (termed Int.sub.N and Int.sub.C, respectively)
fuse, the split intein mediates PTS, resulting in the generation of
a fusion protein with the intein being spliced out. Plasmids
expressing the fusion proteins SpCas9.sub.N-Int.sub.N and
Int.sub.C-SpCas9.sub.C-VP2 (pU1a-Cas9.sub.n-Int.sub.n and
Int.sub.cCas9.sub.c-( )-VP2, respectively) were generated. The
designation "( )" in the Int.sub.c-Cas9.sub.c plasmid represents
the presence of a linker sequence (e.g., GS, GGGGSx3 or EAAAKx3).
Results show productive intein-mediated reconstitution of
SpCas9-VP2 protein in HEK293 cells by co-transfection (FIG. 5).
Importantly, co-transfection of plasmids expressing
SpCas9.sub.N-Int.sub.N and Int.sub.C-SpCas9.sub.C-VP2 in HEK293
cells led to gene editing based on the EGFP-ON reporter assay, as
shown in FIG. 6. EGFP fluorescence reports Cas9 cleavage and NHEJ
repair and mCherry is constitutively expressed as a control.
The Int.sub.C-SpCas9.sub.C protein to be grafted on VP2 is equal or
smaller than EGFP. Since EGFP-AAV2 successfully produced, rAAV
packaging of the Int.sub.C-SpCas9.sub.C-VP2 was investigated.
Guided by structural analysis, SpCas9 split sites close to the
C-terminus of SpCas9 were strategically screened and identified.
Cells were transfected with a plasmid encoding VP1 and VP3
proteins, and a second plasmid encoding Int.sub.c-Cas9.sub.c-(
)-VP2. Results indicate successful incorporation of
Int.sub.c-SpCas9.sub.c-VP2 polypeptide onto rAAV2 capsid (FIG.
7).
TABLE-US-00001 SEQUENCES >SEQ ID NO: 1 SpCas9 nucleic acid
sequence ATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTC
GAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTG
GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTG
CTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTG
TTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG
AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA
CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT
GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA
CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT
GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA
CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA
CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC
GAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC
AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG
AAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCA
ACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGG
ACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG
CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACAT
CCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA
GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA
GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTA
CGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA
GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAG
AGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCA
GATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCA
TTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCC
TACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGA
AAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC
GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA
ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATA
ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC
TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGA
AAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCG
ACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA
CCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA
CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGA
GATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGAT
GAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGC
TGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGA
AGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC
TGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC
TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCC
TGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGC
CCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGA
CAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCT
GGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGA
GAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA
ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGC
TTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAAC
CGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA
CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAA
TCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT
CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT
GGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT
GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAG
TTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGA
ACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGT
TCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG
AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGA
ACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCT
GATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTT
TGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC
CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAG
CGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTT
CGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGG
CAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGA
AAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAA
AGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCT
GGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA
ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA
TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGA
ACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAA
GAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAA
GCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTAC
CCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGAC
CGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAG
AGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC
AGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGC >SEQ ID NO: 2 SpCas9 amino
acid sequence
MYPYDVPDYASPKKKRKVEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKA
ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR
KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI
LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKUNGIRD
KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE
GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN
LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT
LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY
EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSPKKKRKVEAS >SEQ ID NO: 3 SpCas9-VP2 fusion nucleic acid
ATGTACCCATACGATGTTCCAGATTACGCTTCGCCGAAGAAAAAGCGCAAGGTC
GAAGCGTCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTG
GGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTG
CTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTG
TTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAG
AAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAA
CGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCT
GGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGA
CGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACT
GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCA
CATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAA
CAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC
GAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCC
AGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAG
AAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCA
ACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGG
ACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACG
CCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACAT
CCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAA
GAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCA
GCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTA
CGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAA
GCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAG
AGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCA
GATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCA
TTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCC
TACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGA
AAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGC
GCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCA
ACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATA
ACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC
TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGA
AAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCG
ACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATA
CCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAA
CGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGA
GATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGAT
GAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGC
TGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGA
AGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCC
TGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCC
TGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCC
TGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGC
CCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGA
CAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCT
GGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGA
GAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA
ACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGC
TTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAAC
CGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAA
CTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAA
TCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCAT
CAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCT
GGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGT
GAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAG
TTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGA
ACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGT
TCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCG
AGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGA
ACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCT
GATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTT
TGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGAC
CGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAG
CGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTT
CGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGG
CAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGA
AAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAA
AGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCT
GGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAA
ACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTA
TGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGA
ACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAA
GAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAA
GCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTAC
CCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGAC
CGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAG
AGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGAC
AGCCCCAAGAAGAAGAGAAAGGTGGAGGCCAGCGAATTGGCTCCGGGAAAAAA
GAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAA
GGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGC
AGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGT
CTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAAC
GAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACA
TGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACC
TACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGAC
AATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCC
ACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATT
CCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACG
CAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTG
TTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGAT
GCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCAC
CCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTAC
TTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGA
GGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATG
AATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTG
GAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCG
GGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCA
AAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAG
TACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGC
CACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGA
AGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACG
AAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTAT
CTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACAC
AAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGC
CCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCAT
GGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCG
GTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCAC
ACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGA
AAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTC
TGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCC
ATTGGCACCAGATACCTGACTCGTAATCTGTAA >SEQ ID NO: 4 SpCas9-VP2
fusion protein
MYPYDVPDYASPKKKRKVEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINTASGVDAKA
ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR
KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI
LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE
GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN
LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT
LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY
EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK
PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI
DLSQLGGDSPKKKRKVEASELAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFG
QTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWH
CDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFN
RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQV
FTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP
SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQ
SRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGR
DSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPV
ATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDG
HFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWE
LQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL >SEQ ID NO: 5
Linker sequence GGGS >SEQ ID NO: 6 Cas9.sub.n-Int.sub.n
MYPYDVPDYASPKKKRKVEASDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLG
NTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVD
DSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLR
LIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINTASGVDAKA
ILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD
TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDE
HHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKI
LTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK
NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNR
KVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDI
LEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD
KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG
SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE
GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP
QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN
LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVIT
LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY
KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEI
VWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP
KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAK
GYKEVKKDLIIKLPKYSLFELENGRKCLSYETEILTVEYGLLPIGKIVEKRIECTVYSV
DNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFER
ELDLMRVDNLPN >SEQ ID NO: 7 Int.sub.c-Cas9.sub.c-GS-VP2
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCMLASAGELQKGNELALPSK
YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
HQSITGLYETRIDLSQLGGDSPKKKRKVEASGSAPGKKRPVEHSPVEPDSSSGTGKAG
QQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGA
DGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYF
GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGT
TTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQA
VGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYY
LSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYS
WTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKV
MITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVY
LQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQ
YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTR YLTRNL
>SEQ ID NO: 8 Int.sub.c-Cas9.sub.c-GGGGSGGGGSGGGGS-VP2
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCMLASAGELQKGNELALPSK
YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
HQSITGLYETRIDLSQLGGDSPKKKRKVEASGGGGSGGGGSGGGGSAPGKKRPVEHS
PVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMAT
GSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQI
SSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLF
NIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP
QYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSL
DRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQR
VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFG
KQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQG
VLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPAN
PSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTV
DTNGVYSEPRPIGTRYLTRNL >SEQ ID NO: 9
Int.sub.c-Cas9.sub.c-EAAAKEAAAKEAAAK-VP2
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCMLASAGELQKGNELALPSK
YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDK
VLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLI
HQSITGLYETRIDLSQLGGDSPKKKRKVEASEAAAKEAAAKEAAAKAPGKKRPVEH
SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMAT
GSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQI
SSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLF
NIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVP
QYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSL
DRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQR
VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFG
KQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQG
VLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPAN
PSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTV
DTNGVYSEPRPIGTRYLTRNL >SEQ ID NO: 10 pU1a-Cas9.sub.c-Int.sub.c
CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCG
GGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAG
AGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCTCTAGAATGGAGGCG
GTACTATGTAGATGAGAATTCAGGAGCAAACTGGGAAAAGCAACTGCTTCCAAA
TATTTGTGATTTTTACAGTGTAGTTTTGGAAAAACTCTTAGCCTACCAATTCTTCT
AAGTGTTTTAAAATGTGGGAGCCAGTACACATGAAGTTATAGAGTGTTTTAATGA
GGCTTAAATATTTACCGTAACTATGAAATGCTACGCATATCATGCTGTTCAGGCT
CCGTGGCCACGCAACTCATACTACCGGTGCCACCATGTACCCATACGATGTTCCA
GATTACGCTTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCGACAAGAAGTAC
AGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGAC
GAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCAC
AGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCC
GAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAA
CCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGA
CAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCA
CGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAA
GTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGC
CGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC
TTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTC
ATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCC
AGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGG
CTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGC
AACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACC
TGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGG
ACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAA
GAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGAT
CACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCA
GGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAA
AGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGG
AGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGA
CGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCA
GCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCA
CGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGA
AAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCC
AGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCAC
CCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCAT
CGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAA
GCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAA
ATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAA
GGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCT
GAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGG
CGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATT
ATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGAT
ATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTG
AAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGG
AGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGAC
AAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAAC
AGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATC
CAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAAT
CTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTG
GACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAA
ATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAG
AATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAG
AACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACC
TGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGT
CCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCAT
CGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACG
TGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGA
ACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAG
GCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAA
CCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTA
AGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGT
CCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGAT
CAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGC
CCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAA
GGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGG
CTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGAT
TACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGA
AACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGT
GCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGG
CTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAG
AAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGC
CTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAA
GAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAA
GAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCT
GATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGTGT
CTGTCGTATGAGACCGAGATCCTGACCGTGGAGTATGGACTGCTGCCGATTGGAA
AGATTGTGGAGAAGCGCATTGAGTGCACCGTGTACAGCGTGGATAACAATGGCA
ACATCTATACACAGCCAGTGGCCCAGTGGCACGACCGCGGAGAGCAGGAGGTCT
TCGAGTACTGCCTGGAGGATGGCAGCCTGATTCGCGCCACCAAGGATCATAAGTT
CATGACGGTGGACGGACAGATGCTGCCCATCGATGAGATTTTTGAGCGCGAGCT
GGATCTGATGCGCGTGGATAACCTGCCGAATTAAGAATTCGATCTTTTTCCCTCT
GCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAAT
AAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTC
GGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCG
CTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG
GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG >SEQ ID NO: 11
Int.sub.c-Cas9.sub.c-GS-VP2
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA
TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA
CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA
GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAAC
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC
TGGCTAGCGCCACCATGATCAAGATTGCCACGCGCAAGTACCTGGGCAAGCAGA
ACGTGTACGACATCGGAGTGGAGCGCGATCACAACTTTGCCCTGAAGAATGGCT
TTATTGCCTCGAACTGTATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA
ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG
AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGA
GTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC
GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA
CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA
GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT
CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCC
CAAGAAGAAGAGAAAGGTGGAGGCCAGCGGATCCGCTCCGGGAAAAAAGAGGC
CGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGG
GCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACT
CAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGG
AACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGG
CGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATG
GGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAAC
AACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCAC
TACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCC
ACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACC
CAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAA
TGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACT
GACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCC
CGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAA
CAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCT
TCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACG
TTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCC
TCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACC
ACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACC
AGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGAC
ATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCA
CCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAA
GGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAA
GGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAG
GAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCA
ACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCG
TTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTG
GGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGA
TTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTG
CGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTA
CTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACA
GCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAA
TGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGC
ACCAGATACCTGACTCGTAATCTGTAAGAATTAAACCCGCTGATCAGCCTCGACT
GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGAC
CCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCG
CATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCA
AGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA TGG >SEQ
ID NO: 12 Int.sub.c-Cas9.sub.c-GGGGSx3-VP2
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA
TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA
CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA
GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAAC
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC
TGGCTAGCGCCACCATGATCAAGATTGCCACGCGCAAGTACCTGGGCAAGCAGA
ACGTGTACGACATCGGAGTGGAGCGCGATCACAACTTTGCCCTGAAGAATGGCT
TTATTGCCTCGAACTGTATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA
ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG
AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGA
GTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC
GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA
CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA
GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT
CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCC
CAAGAAGAAGAGAAAGGTGGAGGCCAGCGGTGGCGGCGGTTCAGGCGGAGGTG
GCTCTGGGGGCGGGGGTTCTGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTC
CTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAA
GAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCC
AGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGC
TACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGG
GTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCAT
CACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAA
ACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGC
ACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGA
CTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTT
CAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGAC
GATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAG
CTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAG
ACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGC
AGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTA
CCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAG
CTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTAC
CTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGG
CTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGC
TTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAA
CAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGA
CTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAA
GTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACA
AATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACC
AATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCA
ACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGG
TCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCAC
ACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACA
CCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACC
ACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGG
TCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAAT
CCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTG
TGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGAC
TCGTAATCTGTAAGAATTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTG
CCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCA
CTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAG
GTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG
GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG >SEQ ID NO: 13
Int.sub.c-Cas9.sub.c-EAAAKx3-VP2
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTG
GCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT
AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAA
ACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTG
ACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATG
GGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGA
TGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT
TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAA
CGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA
GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAAC
CCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGC
TGGCTAGCGCCACCATGATCAAGATTGCCACGCGCAAGTACCTGGGCAAGCAGA
ACGTGTACGACATCGGAGTGGAGCGCGATCACAACTTTGCCCTGAAGAATGGCT
TTATTGCCTCGAACTGTATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGA
ACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAG
AAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAG
CACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGA
GTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC
GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGA
CCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAA
GAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCAT
CACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCCC
CAAGAAGAAGAGAAAGGTGGAGGCCAGCGAGGCAGCAGCCAAAGAGGCCGCTG
CCAAGGAGGCAGCGGCTAAAGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCT
CCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCA
AGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCC
CAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGG
CTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTG
GGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTC
ATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACA
AACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAG
CACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTG
ACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACT
TCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGA
CGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCA
GCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCA
GACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAG
GCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCG
TACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGC
AGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGT
ACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAA
GGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTG
GCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAAC
AACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGA
GACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAA
AAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAA
CAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAA
CCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGG
CAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCAT
GGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCC
ACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAA
CACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGA
CCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACA
GGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGA
ATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTAC
TGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTG
ACTCGTAATCTGTAAGAATTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGT
TGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC
CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGT
AGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGAT
TGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG
This disclosure is not limited in its application to the details of
construction and the arrangement of components set forth in this
description or illustrated in the drawings. The disclosure is
capable of other embodiments and of being practiced or of being
carried out in various ways. Also, the phraseology and terminology
used herein is for the purpose of description and should not be
regarded as limiting. The use of "including," "comprising," or
"having," "containing," "involving," and variations thereof herein,
is meant to encompass the items listed thereafter and equivalents
thereof as well as additional items.
Having thus described several aspects of at least one embodiment of
this disclosure, it is to be appreciated various alterations,
modifications, and improvements will readily occur to those skilled
in the art. Such alterations, modifications, and improvements are
intended to be part of this disclosure, and are intended to be
within the spirit and scope of the disclosure. Accordingly, the
foregoing description and drawings are by way of example only.
SEQUENCE LISTINGS
1
1314197DNAArtificial SequenceSynthetic Polynucleotide 1atgtacccat
acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg 60tccgacaaga
agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
120atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
caccgaccgg 180cacagcatca agaagaacct gatcggagcc ctgctgttcg
acagcggcga aacagccgag 240gccacccggc tgaagagaac cgccagaaga
agatacacca gacggaagaa ccggatctgc 300tatctgcaag agatcttcag
caacgagatg gccaaggtgg acgacagctt cttccacaga 360ctggaagagt
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
420aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
cctgagaaag 480aaactggtgg acagcaccga caaggccgac ctgcggctga
tctatctggc cctggcccac 540atgatcaagt tccggggcca cttcctgatc
gagggcgacc tgaaccccga caacagcgac 600gtggacaagc tgttcatcca
gctggtgcag acctacaacc agctgttcga ggaaaacccc 660atcaacgcca
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
720cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
gttcggcaac 780ctgattgccc tgagcctggg cctgaccccc aacttcaaga
gcaacttcga cctggccgag 840gatgccaaac tgcagctgag caaggacacc
tacgacgacg acctggacaa cctgctggcc 900cagatcggcg accagtacgc
cgacctgttt ctggccgcca agaacctgtc cgacgccatc 960ctgctgagcg
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
1020atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
tctcgtgcgg 1080cagcagctgc ctgagaagta caaagagatt ttcttcgacc
agagcaagaa cggctacgcc 1140ggctacattg acggcggagc cagccaggaa
gagttctaca agttcatcaa gcccatcctg 1200gaaaagatgg acggcaccga
ggaactgctc gtgaagctga acagagagga cctgctgcgg 1260aagcagcgga
ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
1320gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
ggaaaagatc 1380gagaagatcc tgaccttccg catcccctac tacgtgggcc
ctctggccag gggaaacagc 1440agattcgcct ggatgaccag aaagagcgag
gaaaccatca ccccctggaa cttcgaggaa 1500gtggtggaca agggcgcttc
cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1560aacctgccca
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
1620tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
cgccttcctg 1680agcggcgagc agaaaaaggc catcgtggac ctgctgttca
agaccaaccg gaaagtgacc 1740gtgaagcagc tgaaagagga ctacttcaag
aaaatcgagt gcttcgactc cgtggaaatc 1800tccggcgtgg aagatcggtt
caacgcctcc ctgggcacat accacgatct gctgaaaatt 1860atcaaggaca
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
1920ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
aacctatgcc 1980cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
ggagatacac cggctggggc 2040aggctgagcc ggaagctgat caacggcatc
cgggacaagc agtccggcaa gacaatcctg 2100gatttcctga agtccgacgg
cttcgccaac agaaacttca tgcagctgat ccacgacgac 2160agcctgacct
ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg
2220cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
cctgcagaca 2280gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc
acaagcccga gaacatcgtg 2340atcgaaatgg ccagagagaa ccagaccacc
cagaagggac agaagaacag ccgcgagaga 2400atgaagcgga tcgaagaggg
catcaaagag ctgggcagcc agatcctgaa agaacacccc 2460gtggaaaaca
cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg
2520gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
tgtggaccat 2580atcgtgcctc agagctttct gaaggacgac tccatcgaca
acaaggtgct gaccagaagc 2640gacaagaacc ggggcaagag cgacaacgtg
ccctccgaag aggtcgtgaa gaagatgaag 2700aactactggc ggcagctgct
gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2760accaaggccg
agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag
2820ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
ccggatgaac 2880actaagtacg acgagaatga caagctgatc cgggaagtga
aagtgatcac cctgaagtcc 2940aagctggtgt ccgatttccg gaaggatttc
cagttttaca aagtgcgcga gatcaacaac 3000taccaccacg cccacgacgc
ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3060taccctaagc
tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag
3120atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
cttctacagc 3180aacatcatga actttttcaa gaccgagatt accctggcca
acggcgagat ccggaagcgg 3240cctctgatcg agacaaacgg cgaaaccggg
gagatcgtgt gggataaggg ccgggatttt 3300gccaccgtgc ggaaagtgct
gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3360cagacaggcg
gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc
3420gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
caccgtggcc 3480tattctgtgc tggtggtggc caaagtggaa aagggcaagt
ccaagaaact gaagagtgtg 3540aaagagctgc tggggatcac catcatggaa
agaagcagct tcgagaagaa tcccatcgac 3600tttctggaag ccaagggcta
caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3660tactccctgt
tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgaactg
3720cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
cctggccagc 3780cactatgaga agctgaaggg ctcccccgag gataatgagc
agaaacagct gtttgtggaa 3840cagcacaagc actacctgga cgagatcatc
gagcagatca gcgagttctc caagagagtg 3900atcctggccg acgctaatct
ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3960cccatcagag
agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc
4020cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
cagcaccaaa 4080gaggtgctgg acgccaccct gatccaccag agcatcaccg
gcctgtacga gacacggatc 4140gacctgtctc agctgggagg cgacagcccc
aagaagaaga gaaaggtgga ggccagc 419721399PRTArtificial
SequenceSynthetic Polypeptide 2Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ser Pro Lys Lys Lys Arg1 5 10 15Lys Val Glu Ala Ser Asp Lys Lys
Tyr Ser Ile Gly Leu Asp Ile Gly 20 25 30Thr Asn Ser Val Gly Trp Ala
Val Ile Thr Asp Glu Tyr Lys Val Pro 35 40 45Ser Lys Lys Phe Lys Val
Leu Gly Asn Thr Asp Arg His Ser Ile Lys 50 55 60Lys Asn Leu Ile Gly
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu65 70 75 80Ala Thr Arg
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys 85 90 95Asn Arg
Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys 100 105
110Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
115 120 125Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
Val Asp 130 135 140Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
His Leu Arg Lys145 150 155 160Lys Leu Val Asp Ser Thr Asp Lys Ala
Asp Leu Arg Leu Ile Tyr Leu 165 170 175Ala Leu Ala His Met Ile Lys
Phe Arg Gly His Phe Leu Ile Glu Gly 180 185 190Asp Leu Asn Pro Asp
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu 195 200 205Val Gln Thr
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser 210 215 220Gly
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg225 230
235 240Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn
Gly 245 250 255Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr
Pro Asn Phe 260 265 270Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys
Leu Gln Leu Ser Lys 275 280 285Asp Thr Tyr Asp Asp Asp Leu Asp Asn
Leu Leu Ala Gln Ile Gly Asp 290 295 300Gln Tyr Ala Asp Leu Phe Leu
Ala Ala Lys Asn Leu Ser Asp Ala Ile305 310 315 320Leu Leu Ser Asp
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro 325 330 335Leu Ser
Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu 340 345
350Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
355 360 365Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
Ile Asp 370 375 380Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
Lys Pro Ile Leu385 390 395 400Glu Lys Met Asp Gly Thr Glu Glu Leu
Leu Val Lys Leu Asn Arg Glu 405 410 415Asp Leu Leu Arg Lys Gln Arg
Thr Phe Asp Asn Gly Ser Ile Pro His 420 425 430Gln Ile His Leu Gly
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp 435 440 445Phe Tyr Pro
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu 450 455 460Thr
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser465 470
475 480Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro
Trp 485 490 495Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln
Ser Phe Ile 500 505 510Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro
Asn Glu Lys Val Leu 515 520 525Pro Lys His Ser Leu Leu Tyr Glu Tyr
Phe Thr Val Tyr Asn Glu Leu 530 535 540Thr Lys Val Lys Tyr Val Thr
Glu Gly Met Arg Lys Pro Ala Phe Leu545 550 555 560Ser Gly Glu Gln
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn 565 570 575Arg Lys
Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile 580 585
590Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
595 600 605Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
Asp Lys 610 615 620Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
Glu Asp Ile Val625 630 635 640Leu Thr Leu Thr Leu Phe Glu Asp Arg
Glu Met Ile Glu Glu Arg Leu 645 650 655Lys Thr Tyr Ala His Leu Phe
Asp Asp Lys Val Met Lys Gln Leu Lys 660 665 670Arg Arg Arg Tyr Thr
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn 675 680 685Gly Ile Arg
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys 690 695 700Ser
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp705 710
715 720Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly
Gln 725 730 735Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
Ser Pro Ala 740 745 750Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val
Val Asp Glu Leu Val 755 760 765Lys Val Met Gly Arg His Lys Pro Glu
Asn Ile Val Ile Glu Met Ala 770 775 780Arg Glu Asn Gln Thr Thr Gln
Lys Gly Gln Lys Asn Ser Arg Glu Arg785 790 795 800Met Lys Arg Ile
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu 805 810 815Lys Glu
His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr 820 825
830Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu
835 840 845Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val
Pro Gln 850 855 860Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
Leu Thr Arg Ser865 870 875 880Asp Lys Asn Arg Gly Lys Ser Asp Asn
Val Pro Ser Glu Glu Val Val 885 890 895Lys Lys Met Lys Asn Tyr Trp
Arg Gln Leu Leu Asn Ala Lys Leu Ile 900 905 910Thr Gln Arg Lys Phe
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu 915 920 925Ser Glu Leu
Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr 930 935 940Arg
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn945 950
955 960Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
Ile 965 970 975Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
Phe Gln Phe 980 985 990Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His
Ala His Asp Ala Tyr 995 1000 1005Leu Asn Ala Val Val Gly Thr Ala
Leu Ile Lys Lys Tyr Pro Lys 1010 1015 1020Leu Glu Ser Glu Phe Val
Tyr Gly Asp Tyr Lys Val Tyr Asp Val 1025 1030 1035Arg Lys Met Ile
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr 1040 1045 1050Ala Lys
Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1055 1060
1065Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile
1070 1075 1080Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys
Gly Arg 1085 1090 1095Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
Pro Gln Val Asn 1100 1105 1110Ile Val Lys Lys Thr Glu Val Gln Thr
Gly Gly Phe Ser Lys Glu 1115 1120 1125Ser Ile Leu Pro Lys Arg Asn
Ser Asp Lys Leu Ile Ala Arg Lys 1130 1135 1140Lys Asp Trp Asp Pro
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr 1145 1150 1155Val Ala Tyr
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys 1160 1165 1170Ser
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1175 1180
1185Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu
1190 1195 1200Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile
Lys Leu 1205 1210 1215Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
Arg Lys Arg Met 1220 1225 1230Leu Ala Ser Ala Gly Glu Leu Gln Lys
Gly Asn Glu Leu Ala Leu 1235 1240 1245Pro Ser Lys Tyr Val Asn Phe
Leu Tyr Leu Ala Ser His Tyr Glu 1250 1255 1260Lys Leu Lys Gly Ser
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe 1265 1270 1275Val Glu Gln
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile 1280 1285 1290Ser
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp 1295 1300
1305Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg
1310 1315 1320Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr
Asn Leu 1325 1330 1335Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
Thr Ile Asp Arg 1340 1345 1350Lys Arg Tyr Thr Ser Thr Lys Glu Val
Leu Asp Ala Thr Leu Ile 1355 1360 1365His Gln Ser Ile Thr Gly Leu
Tyr Glu Thr Arg Ile Asp Leu Ser 1370 1375 1380Gln Leu Gly Gly Asp
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala 1385 1390
1395Ser35997DNAArtificial SequenceSynthetic Polynucleotide
3atgtacccat acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg
60tccgacaaga agtacagcat cggcctggac atcggcacca actctgtggg ctgggccgtg
120atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
caccgaccgg 180cacagcatca agaagaacct gatcggagcc ctgctgttcg
acagcggcga aacagccgag 240gccacccggc tgaagagaac cgccagaaga
agatacacca gacggaagaa ccggatctgc 300tatctgcaag agatcttcag
caacgagatg gccaaggtgg acgacagctt cttccacaga 360ctggaagagt
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc
420aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
cctgagaaag 480aaactggtgg acagcaccga caaggccgac ctgcggctga
tctatctggc cctggcccac 540atgatcaagt tccggggcca cttcctgatc
gagggcgacc tgaaccccga caacagcgac 600gtggacaagc tgttcatcca
gctggtgcag acctacaacc agctgttcga ggaaaacccc 660atcaacgcca
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga
720cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
gttcggcaac 780ctgattgccc tgagcctggg cctgaccccc aacttcaaga
gcaacttcga cctggccgag 840gatgccaaac tgcagctgag caaggacacc
tacgacgacg acctggacaa cctgctggcc 900cagatcggcg accagtacgc
cgacctgttt ctggccgcca agaacctgtc cgacgccatc 960ctgctgagcg
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct
1020atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
tctcgtgcgg 1080cagcagctgc ctgagaagta caaagagatt ttcttcgacc
agagcaagaa cggctacgcc 1140ggctacattg acggcggagc cagccaggaa
gagttctaca agttcatcaa gcccatcctg 1200gaaaagatgg acggcaccga
ggaactgctc gtgaagctga acagagagga cctgctgcgg 1260aagcagcgga
ccttcgacaa cggcagcatc ccccaccaga tccacctggg agagctgcac
1320gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
ggaaaagatc 1380gagaagatcc tgaccttccg catcccctac tacgtgggcc
ctctggccag gggaaacagc 1440agattcgcct ggatgaccag aaagagcgag
gaaaccatca ccccctggaa cttcgaggaa 1500gtggtggaca agggcgcttc
cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1560aacctgccca
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg
1620tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
cgccttcctg 1680agcggcgagc agaaaaaggc catcgtggac ctgctgttca
agaccaaccg gaaagtgacc 1740gtgaagcagc tgaaagagga ctacttcaag
aaaatcgagt gcttcgactc cgtggaaatc 1800tccggcgtgg aagatcggtt
caacgcctcc ctgggcacat accacgatct gctgaaaatt 1860atcaaggaca
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg
1920ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
aacctatgcc 1980cacctgttcg acgacaaagt gatgaagcag ctgaagcggc
ggagatacac cggctggggc 2040aggctgagcc ggaagctgat caacggcatc
cgggacaagc agtccggcaa gacaatcctg 2100gatttcctga agtccgacgg
cttcgccaac agaaacttca tgcagctgat ccacgacgac
2160agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
cgatagcctg 2220cacgagcaca ttgccaatct ggccggcagc cccgccatta
agaagggcat cctgcagaca 2280gtgaaggtgg tggacgagct cgtgaaagtg
atgggccggc acaagcccga gaacatcgtg 2340atcgaaatgg ccagagagaa
ccagaccacc cagaagggac agaagaacag ccgcgagaga 2400atgaagcgga
tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc
2460gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
gaatgggcgg 2520gatatgtacg tggaccagga actggacatc aaccggctgt
ccgactacga tgtggaccat 2580atcgtgcctc agagctttct gaaggacgac
tccatcgaca acaaggtgct gaccagaagc 2640gacaagaacc ggggcaagag
cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2700aactactggc
ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg
2760accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
caagagacag 2820ctggtggaaa cccggcagat cacaaagcac gtggcacaga
tcctggactc ccggatgaac 2880actaagtacg acgagaatga caagctgatc
cgggaagtga aagtgatcac cctgaagtcc 2940aagctggtgt ccgatttccg
gaaggatttc cagttttaca aagtgcgcga gatcaacaac 3000taccaccacg
cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag
3060taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
cgtgcggaag 3120atgatcgcca agagcgagca ggaaatcggc aaggctaccg
ccaagtactt cttctacagc 3180aacatcatga actttttcaa gaccgagatt
accctggcca acggcgagat ccggaagcgg 3240cctctgatcg agacaaacgg
cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3300gccaccgtgc
ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg
3360cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
taagctgatc 3420gccagaaaga aggactggga ccctaagaag tacggcggct
tcgacagccc caccgtggcc 3480tattctgtgc tggtggtggc caaagtggaa
aagggcaagt ccaagaaact gaagagtgtg 3540aaagagctgc tggggatcac
catcatggaa agaagcagct tcgagaagaa tcccatcgac 3600tttctggaag
ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag
3660tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
cggcgaactg 3720cagaagggaa acgaactggc cctgccctcc aaatatgtga
acttcctgta cctggccagc 3780cactatgaga agctgaaggg ctcccccgag
gataatgagc agaaacagct gtttgtggaa 3840cagcacaagc actacctgga
cgagatcatc gagcagatca gcgagttctc caagagagtg 3900atcctggccg
acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag
3960cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
tctgggagcc 4020cctgccgcct tcaagtactt tgacaccacc atcgaccgga
agaggtacac cagcaccaaa 4080gaggtgctgg acgccaccct gatccaccag
agcatcaccg gcctgtacga gacacggatc 4140gacctgtctc agctgggagg
cgacagcccc aagaagaaga gaaaggtgga ggccagcgaa 4200ttggctccgg
gaaaaaagag gccggtagag cactctcctg tggagccaga ctcctcctcg
4260ggaaccggaa aggcgggcca gcagcctgca agaaaaagat tgaattttgg
tcagactgga 4320gacgcagact cagtacctga cccccagcct ctcggacagc
caccagcagc cccctctggt 4380ctgggaacta atacgatggc tacaggcagt
ggcgcaccaa tggcagacaa taacgagggc 4440gccgacggag tgggtaattc
ctcgggaaat tggcattgcg attccacatg gatgggcgac 4500agagtcatca
ccaccagcac ccgaacctgg gccctgccca cctacaacaa ccacctctac
4560aaacaaattt ccagccaatc aggagcctcg aacgacaatc actactttgg
ctacagcacc 4620ccttgggggt attttgactt caacagattc cactgccact
tttcaccacg tgactggcaa 4680agactcatca acaacaactg gggattccga
cccaagagac tcaacttcaa gctctttaac 4740attcaagtca aagaggtcac
gcagaatgac ggtacgacga cgattgccaa taaccttacc 4800agcacggttc
aggtgtttac tgactcggag taccagctcc cgtacgtcct cggctcggcg
4860catcaaggat gcctcccgcc gttcccagca gacgtcttca tggtgccaca
gtatggatac 4920ctcaccctga acaacgggag tcaggcagta ggacgctctt
cattttactg cctggagtac 4980tttccttctc agatgctgcg taccggaaac
aactttacct tcagctacac ttttgaggac 5040gttcctttcc acagcagcta
cgctcacagc cagagtctgg accgtctcat gaatcctctc 5100atcgaccagt
acctgtatta cttgagcaga acaaacactc caagtggaac caccacgcag
5160tcaaggcttc agttttctca ggccggagcg agtgacattc gggaccagtc
taggaactgg 5220cttcctggac cctgttaccg ccagcagcga gtatcaaaga
catctgcgga taacaacaac 5280agtgaatact cgtggactgg agctaccaag
taccacctca atggcagaga ctctctggtg 5340aatccgggcc cggccatggc
aagccacaag gacgatgaag aaaagttttt tcctcagagc 5400ggggttctca
tctttgggaa gcaaggctca gagaaaacaa atgtggacat tgaaaaggtc
5460atgattacag acgaagagga aatcaggaca accaatcccg tggctacgga
gcagtatggt 5520tctgtatcta ccaacctcca gagaggcaac agacaagcag
ctaccgcaga tgtcaacaca 5580caaggcgttc ttccaggcat ggtctggcag
gacagagatg tgtaccttca ggggcccatc 5640tgggcaaaga ttccacacac
ggacggacat tttcacccct ctcccctcat gggtggattc 5700ggacttaaac
accctcctcc acagattctc atcaagaaca ccccggtacc tgcgaatcct
5760tcgaccacct tcagtgcggc aaagtttgct tccttcatca cacagtactc
cacgggacag 5820gtcagcgtgg agatcgagtg ggagctgcag aaggaaaaca
gcaaacgctg gaatcccgaa 5880attcagtaca cttccaacta caacaagtct
gttaatgtgg actttactgt ggacactaat 5940ggcgtgtatt cagagcctcg
ccccattggc accagatacc tgactcgtaa tctgtaa 599741998PRTArtificial
SequenceSynthetic Polypeptide 4Met Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ser Pro Lys Lys Lys Arg1 5 10 15Lys Val Glu Ala Ser Asp Lys Lys
Tyr Ser Ile Gly Leu Asp Ile Gly 20 25 30Thr Asn Ser Val Gly Trp Ala
Val Ile Thr Asp Glu Tyr Lys Val Pro 35 40 45Ser Lys Lys Phe Lys Val
Leu Gly Asn Thr Asp Arg His Ser Ile Lys 50 55 60Lys Asn Leu Ile Gly
Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu65 70 75 80Ala Thr Arg
Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys 85 90 95Asn Arg
Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys 100 105
110Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu
115 120 125Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile
Val Asp 130 135 140Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr
His Leu Arg Lys145 150 155 160Lys Leu Val Asp Ser Thr Asp Lys Ala
Asp Leu Arg Leu Ile Tyr Leu 165 170 175Ala Leu Ala His Met Ile Lys
Phe Arg Gly His Phe Leu Ile Glu Gly 180 185 190Asp Leu Asn Pro Asp
Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu 195 200 205Val Gln Thr
Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser 210 215 220Gly
Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg225 230
235 240Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn
Gly 245 250 255Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr
Pro Asn Phe 260 265 270Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys
Leu Gln Leu Ser Lys 275 280 285Asp Thr Tyr Asp Asp Asp Leu Asp Asn
Leu Leu Ala Gln Ile Gly Asp 290 295 300Gln Tyr Ala Asp Leu Phe Leu
Ala Ala Lys Asn Leu Ser Asp Ala Ile305 310 315 320Leu Leu Ser Asp
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro 325 330 335Leu Ser
Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu 340 345
350Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys
355 360 365Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr
Ile Asp 370 375 380Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile
Lys Pro Ile Leu385 390 395 400Glu Lys Met Asp Gly Thr Glu Glu Leu
Leu Val Lys Leu Asn Arg Glu 405 410 415Asp Leu Leu Arg Lys Gln Arg
Thr Phe Asp Asn Gly Ser Ile Pro His 420 425 430Gln Ile His Leu Gly
Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp 435 440 445Phe Tyr Pro
Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu 450 455 460Thr
Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser465 470
475 480Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro
Trp 485 490 495Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln
Ser Phe Ile 500 505 510Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro
Asn Glu Lys Val Leu 515 520 525Pro Lys His Ser Leu Leu Tyr Glu Tyr
Phe Thr Val Tyr Asn Glu Leu 530 535 540Thr Lys Val Lys Tyr Val Thr
Glu Gly Met Arg Lys Pro Ala Phe Leu545 550 555 560Ser Gly Glu Gln
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn 565 570 575Arg Lys
Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile 580 585
590Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn
595 600 605Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys
Asp Lys 610 615 620Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu
Glu Asp Ile Val625 630 635 640Leu Thr Leu Thr Leu Phe Glu Asp Arg
Glu Met Ile Glu Glu Arg Leu 645 650 655Lys Thr Tyr Ala His Leu Phe
Asp Asp Lys Val Met Lys Gln Leu Lys 660 665 670Arg Arg Arg Tyr Thr
Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn 675 680 685Gly Ile Arg
Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys 690 695 700Ser
Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp705 710
715 720Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly
Gln 725 730 735Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly
Ser Pro Ala 740 745 750Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val
Val Asp Glu Leu Val 755 760 765Lys Val Met Gly Arg His Lys Pro Glu
Asn Ile Val Ile Glu Met Ala 770 775 780Arg Glu Asn Gln Thr Thr Gln
Lys Gly Gln Lys Asn Ser Arg Glu Arg785 790 795 800Met Lys Arg Ile
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu 805 810 815Lys Glu
His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr 820 825
830Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu
835 840 845Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val
Pro Gln 850 855 860Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val
Leu Thr Arg Ser865 870 875 880Asp Lys Asn Arg Gly Lys Ser Asp Asn
Val Pro Ser Glu Glu Val Val 885 890 895Lys Lys Met Lys Asn Tyr Trp
Arg Gln Leu Leu Asn Ala Lys Leu Ile 900 905 910Thr Gln Arg Lys Phe
Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu 915 920 925Ser Glu Leu
Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr 930 935 940Arg
Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn945 950
955 960Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val
Ile 965 970 975Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp
Phe Gln Phe 980 985 990Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His
Ala His Asp Ala Tyr 995 1000 1005Leu Asn Ala Val Val Gly Thr Ala
Leu Ile Lys Lys Tyr Pro Lys 1010 1015 1020Leu Glu Ser Glu Phe Val
Tyr Gly Asp Tyr Lys Val Tyr Asp Val 1025 1030 1035Arg Lys Met Ile
Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr 1040 1045 1050Ala Lys
Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr 1055 1060
1065Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile
1070 1075 1080Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys
Gly Arg 1085 1090 1095Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met
Pro Gln Val Asn 1100 1105 1110Ile Val Lys Lys Thr Glu Val Gln Thr
Gly Gly Phe Ser Lys Glu 1115 1120 1125Ser Ile Leu Pro Lys Arg Asn
Ser Asp Lys Leu Ile Ala Arg Lys 1130 1135 1140Lys Asp Trp Asp Pro
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr 1145 1150 1155Val Ala Tyr
Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys 1160 1165 1170Ser
Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile 1175 1180
1185Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu
1190 1195 1200Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile
Lys Leu 1205 1210 1215Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly
Arg Lys Arg Met 1220 1225 1230Leu Ala Ser Ala Gly Glu Leu Gln Lys
Gly Asn Glu Leu Ala Leu 1235 1240 1245Pro Ser Lys Tyr Val Asn Phe
Leu Tyr Leu Ala Ser His Tyr Glu 1250 1255 1260Lys Leu Lys Gly Ser
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe 1265 1270 1275Val Glu Gln
His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile 1280 1285 1290Ser
Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp 1295 1300
1305Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg
1310 1315 1320Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr
Asn Leu 1325 1330 1335Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr
Thr Ile Asp Arg 1340 1345 1350Lys Arg Tyr Thr Ser Thr Lys Glu Val
Leu Asp Ala Thr Leu Ile 1355 1360 1365His Gln Ser Ile Thr Gly Leu
Tyr Glu Thr Arg Ile Asp Leu Ser 1370 1375 1380Gln Leu Gly Gly Asp
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala 1385 1390 1395Ser Glu Leu
Ala Pro Gly Lys Lys Arg Pro Val Glu His Ser Pro 1400 1405 1410Val
Glu Pro Asp Ser Ser Ser Gly Thr Gly Lys Ala Gly Gln Gln 1415 1420
1425Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr Gly Asp Ala Asp
1430 1435 1440Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro Ala
Ala Pro 1445 1450 1455Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly
Ser Gly Ala Pro 1460 1465 1470Met Ala Asp Asn Asn Glu Gly Ala Asp
Gly Val Gly Asn Ser Ser 1475 1480 1485Gly Asn Trp His Cys Asp Ser
Thr Trp Met Gly Asp Arg Val Ile 1490 1495 1500Thr Thr Ser Thr Arg
Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 1505 1510 1515Leu Tyr Lys
Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn 1520 1525 1530His
Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 1535 1540
1545Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile
1550 1555 1560Asn Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe
Lys Leu 1565 1570 1575Phe Asn Ile Gln Val Lys Glu Val Thr Gln Asn
Asp Gly Thr Thr 1580 1585 1590Thr Ile Ala Asn Asn Leu Thr Ser Thr
Val Gln Val Phe Thr Asp 1595 1600 1605Ser Glu Tyr Gln Leu Pro Tyr
Val Leu Gly Ser Ala His Gln Gly 1610 1615 1620Cys Leu Pro Pro Phe
Pro Ala Asp Val Phe Met Val Pro Gln Tyr 1625 1630 1635Gly Tyr Leu
Thr Leu Asn Asn Gly Ser Gln Ala Val Gly Arg Ser 1640 1645 1650Ser
Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr 1655 1660
1665Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu Asp Val Pro Phe
1670 1675 1680His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Leu
Met Asn 1685 1690 1695Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser
Arg Thr Asn Thr 1700 1705 1710Pro Ser Gly Thr Thr Thr Gln Ser Arg
Leu Gln Phe Ser Gln Ala 1715 1720 1725Gly Ala Ser Asp Ile Arg Asp
Gln Ser Arg Asn Trp Leu Pro Gly 1730 1735 1740Pro Cys Tyr Arg Gln
Gln Arg Val Ser Lys Thr Ser Ala Asp Asn 1745 1750 1755Asn Asn Ser
Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu 1760 1765 1770Asn
Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser 1775 1780
1785His Lys Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu
1790 1795 1800Ile Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp
Ile Glu 1805 1810 1815Lys Val Met Ile Thr Asp Glu Glu Glu
Ile Arg Thr Thr Asn Pro 1820 1825 1830Val Ala Thr Glu Gln Tyr Gly
Ser Val Ser Thr Asn Leu Gln Arg 1835 1840 1845Gly Asn Arg Gln Ala
Ala Thr Ala Asp Val Asn Thr Gln Gly Val 1850 1855 1860Leu Pro Gly
Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln Gly 1865 1870 1875Pro
Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro 1880 1885
1890Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln
1895 1900 1905Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser
Thr Thr 1910 1915 1920Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr
Gln Tyr Ser Thr 1925 1930 1935Gly Gln Val Ser Val Glu Ile Glu Trp
Glu Leu Gln Lys Glu Asn 1940 1945 1950Ser Lys Arg Trp Asn Pro Glu
Ile Gln Tyr Thr Ser Asn Tyr Asn 1955 1960 1965Lys Ser Val Asn Val
Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 1970 1975 1980Ser Glu Pro
Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 1985 1990
199554PRTArtificial SequenceSynthetic Polypeptide 5Gly Gly Gly
Ser161333PRTArtificial SequenceSynthetic Polypeptide 6Met Tyr Pro
Tyr Asp Val Pro Asp Tyr Ala Ser Pro Lys Lys Lys Arg1 5 10 15Lys Val
Glu Ala Ser Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly 20 25 30Thr
Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro 35 40
45Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys
50 55 60Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala
Glu65 70 75 80Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr
Arg Arg Lys 85 90 95Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn
Glu Met Ala Lys 100 105 110Val Asp Asp Ser Phe Phe His Arg Leu Glu
Glu Ser Phe Leu Val Glu 115 120 125Glu Asp Lys Lys His Glu Arg His
Pro Ile Phe Gly Asn Ile Val Asp 130 135 140Glu Val Ala Tyr His Glu
Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys145 150 155 160Lys Leu Val
Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu 165 170 175Ala
Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly 180 185
190Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu
195 200 205Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn
Ala Ser 210 215 220Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu
Ser Lys Ser Arg225 230 235 240Arg Leu Glu Asn Leu Ile Ala Gln Leu
Pro Gly Glu Lys Lys Asn Gly 245 250 255Leu Phe Gly Asn Leu Ile Ala
Leu Ser Leu Gly Leu Thr Pro Asn Phe 260 265 270Lys Ser Asn Phe Asp
Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys 275 280 285Asp Thr Tyr
Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp 290 295 300Gln
Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile305 310
315 320Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala
Pro 325 330 335Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His
Gln Asp Leu 340 345 350Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu
Pro Glu Lys Tyr Lys 355 360 365Glu Ile Phe Phe Asp Gln Ser Lys Asn
Gly Tyr Ala Gly Tyr Ile Asp 370 375 380Gly Gly Ala Ser Gln Glu Glu
Phe Tyr Lys Phe Ile Lys Pro Ile Leu385 390 395 400Glu Lys Met Asp
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu 405 410 415Asp Leu
Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His 420 425
430Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp
435 440 445Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys
Ile Leu 450 455 460Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala
Arg Gly Asn Ser465 470 475 480Arg Phe Ala Trp Met Thr Arg Lys Ser
Glu Glu Thr Ile Thr Pro Trp 485 490 495Asn Phe Glu Glu Val Val Asp
Lys Gly Ala Ser Ala Gln Ser Phe Ile 500 505 510Glu Arg Met Thr Asn
Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu 515 520 525Pro Lys His
Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu 530 535 540Thr
Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu545 550
555 560Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr
Asn 565 570 575Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe
Lys Lys Ile 580 585 590Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val
Glu Asp Arg Phe Asn 595 600 605Ala Ser Leu Gly Thr Tyr His Asp Leu
Leu Lys Ile Ile Lys Asp Lys 610 615 620Asp Phe Leu Asp Asn Glu Glu
Asn Glu Asp Ile Leu Glu Asp Ile Val625 630 635 640Leu Thr Leu Thr
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu 645 650 655Lys Thr
Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys 660 665
670Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn
675 680 685Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe
Leu Lys 690 695 700Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu
Ile His Asp Asp705 710 715 720Ser Leu Thr Phe Lys Glu Asp Ile Gln
Lys Ala Gln Val Ser Gly Gln 725 730 735Gly Asp Ser Leu His Glu His
Ile Ala Asn Leu Ala Gly Ser Pro Ala 740 745 750Ile Lys Lys Gly Ile
Leu Gln Thr Val Lys Val Val Asp Glu Leu Val 755 760 765Lys Val Met
Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala 770 775 780Arg
Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg785 790
795 800Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile
Leu 805 810 815Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu
Lys Leu Tyr 820 825 830Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr
Val Asp Gln Glu Leu 835 840 845Asp Ile Asn Arg Leu Ser Asp Tyr Asp
Val Asp His Ile Val Pro Gln 850 855 860Ser Phe Leu Lys Asp Asp Ser
Ile Asp Asn Lys Val Leu Thr Arg Ser865 870 875 880Asp Lys Asn Arg
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val 885 890 895Lys Lys
Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile 900 905
910Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu
915 920 925Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val
Glu Thr 930 935 940Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp
Ser Arg Met Asn945 950 955 960Thr Lys Tyr Asp Glu Asn Asp Lys Leu
Ile Arg Glu Val Lys Val Ile 965 970 975Thr Leu Lys Ser Lys Leu Val
Ser Asp Phe Arg Lys Asp Phe Gln Phe 980 985 990Tyr Lys Val Arg Glu
Ile Asn Asn Tyr His His Ala His Asp Ala Tyr 995 1000 1005Leu Asn
Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys 1010 1015
1020Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val
1025 1030 1035Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys
Ala Thr 1040 1045 1050Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn
Phe Phe Lys Thr 1055 1060 1065Glu Ile Thr Leu Ala Asn Gly Glu Ile
Arg Lys Arg Pro Leu Ile 1070 1075 1080Glu Thr Asn Gly Glu Thr Gly
Glu Ile Val Trp Asp Lys Gly Arg 1085 1090 1095Asp Phe Ala Thr Val
Arg Lys Val Leu Ser Met Pro Gln Val Asn 1100 1105 1110Ile Val Lys
Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu 1115 1120 1125Ser
Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys 1130 1135
1140Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr
1145 1150 1155Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys
Gly Lys 1160 1165 1170Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu
Gly Ile Thr Ile 1175 1180 1185Met Glu Arg Ser Ser Phe Glu Lys Asn
Pro Ile Asp Phe Leu Glu 1190 1195 1200Ala Lys Gly Tyr Lys Glu Val
Lys Lys Asp Leu Ile Ile Lys Leu 1205 1210 1215Pro Lys Tyr Ser Leu
Phe Glu Leu Glu Asn Gly Arg Lys Cys Leu 1220 1225 1230Ser Tyr Glu
Thr Glu Ile Leu Thr Val Glu Tyr Gly Leu Leu Pro 1235 1240 1245Ile
Gly Lys Ile Val Glu Lys Arg Ile Glu Cys Thr Val Tyr Ser 1250 1255
1260Val Asp Asn Asn Gly Asn Ile Tyr Thr Gln Pro Val Ala Gln Trp
1265 1270 1275His Asp Arg Gly Glu Gln Glu Val Phe Glu Tyr Cys Leu
Glu Asp 1280 1285 1290Gly Ser Leu Ile Arg Ala Thr Lys Asp His Lys
Phe Met Thr Val 1295 1300 1305Asp Gly Gln Met Leu Pro Ile Asp Glu
Ile Phe Glu Arg Glu Leu 1310 1315 1320Asp Leu Met Arg Val Asp Asn
Leu Pro Asn 1325 13307803PRTArtificial SequenceSynthetic
Polypeptide 7Met Ile Lys Ile Ala Thr Arg Lys Tyr Leu Gly Lys Gln
Asn Val Tyr1 5 10 15Asp Ile Gly Val Glu Arg Asp His Asn Phe Ala Leu
Lys Asn Gly Phe 20 25 30Ile Ala Ser Asn Cys Met Leu Ala Ser Ala Gly
Glu Leu Gln Lys Gly 35 40 45Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
Asn Phe Leu Tyr Leu Ala 50 55 60Ser His Tyr Glu Lys Leu Lys Gly Ser
Pro Glu Asp Asn Glu Gln Lys65 70 75 80Gln Leu Phe Val Glu Gln His
Lys His Tyr Leu Asp Glu Ile Ile Glu 85 90 95Gln Ile Ser Glu Phe Ser
Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 100 105 110Asp Lys Val Leu
Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg 115 120 125Glu Gln
Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 130 135
140Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys
Arg145 150 155 160Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu
Ile His Gln Ser 165 170 175Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp
Leu Ser Gln Leu Gly Gly 180 185 190Asp Ser Pro Lys Lys Lys Arg Lys
Val Glu Ala Ser Gly Ser Ala Pro 195 200 205Gly Lys Lys Arg Pro Val
Glu His Ser Pro Val Glu Pro Asp Ser Ser 210 215 220Ser Gly Thr Gly
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn225 230 235 240Phe
Gly Gln Thr Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu 245 250
255Gly Gln Pro Pro Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala
260 265 270Thr Gly Ser Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala
Asp Gly 275 280 285Val Gly Asn Ser Ser Gly Asn Trp His Cys Asp Ser
Thr Trp Met Gly 290 295 300Asp Arg Val Ile Thr Thr Ser Thr Arg Thr
Trp Ala Leu Pro Thr Tyr305 310 315 320Asn Asn His Leu Tyr Lys Gln
Ile Ser Ser Gln Ser Gly Ala Ser Asn 325 330 335Asp Asn His Tyr Phe
Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe 340 345 350Asn Arg Phe
His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile 355 360 365Asn
Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe 370 375
380Asn Ile Gln Val Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr
Ile385 390 395 400Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr
Asp Ser Glu Tyr 405 410 415Gln Leu Pro Tyr Val Leu Gly Ser Ala His
Gln Gly Cys Leu Pro Pro 420 425 430Phe Pro Ala Asp Val Phe Met Val
Pro Gln Tyr Gly Tyr Leu Thr Leu 435 440 445Asn Asn Gly Ser Gln Ala
Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu 450 455 460Tyr Phe Pro Ser
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser465 470 475 480Tyr
Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln 485 490
495Ser Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr
500 505 510Leu Ser Arg Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser
Arg Leu 515 520 525Gln Phe Ser Gln Ala Gly Ala Ser Asp Ile Arg Asp
Gln Ser Arg Asn 530 535 540Trp Leu Pro Gly Pro Cys Tyr Arg Gln Gln
Arg Val Ser Lys Thr Ser545 550 555 560Ala Asp Asn Asn Asn Ser Glu
Tyr Ser Trp Thr Gly Ala Thr Lys Tyr 565 570 575His Leu Asn Gly Arg
Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala 580 585 590Ser His Lys
Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu 595 600 605Ile
Phe Gly Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys 610 615
620Val Met Ile Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val
Ala625 630 635 640Thr Glu Gln Tyr Gly Ser Val Ser Thr Asn Leu Gln
Arg Gly Asn Arg 645 650 655Gln Ala Ala Thr Ala Asp Val Asn Thr Gln
Gly Val Leu Pro Gly Met 660 665 670Val Trp Gln Asp Arg Asp Val Tyr
Leu Gln Gly Pro Ile Trp Ala Lys 675 680 685Ile Pro His Thr Asp Gly
His Phe His Pro Ser Pro Leu Met Gly Gly 690 695 700Phe Gly Leu Lys
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro705 710 715 720Val
Pro Ala Asn Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser 725 730
735Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp
740 745 750Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile
Gln Tyr 755 760 765Thr Ser Asn Tyr Asn Lys Ser Val Asn Val Asp Phe
Thr Val Asp Thr 770 775 780Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile
Gly Thr Arg Tyr Leu Thr785 790 795 800Arg Asn Leu8816PRTArtificial
SequenceSynthetic Polypeptide 8Met Ile Lys Ile Ala Thr Arg Lys Tyr
Leu Gly Lys Gln Asn Val Tyr1 5 10 15Asp Ile Gly Val Glu Arg Asp His
Asn Phe Ala Leu Lys Asn Gly Phe 20 25 30Ile Ala Ser Asn Cys Met Leu
Ala Ser Ala Gly Glu Leu Gln Lys Gly 35 40 45Asn Glu Leu Ala Leu Pro
Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 50 55 60Ser His Tyr Glu Lys
Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys65 70 75 80Gln Leu Phe
Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 85 90 95Gln Ile
Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu 100 105
110Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg
115 120
125Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly
130 135 140Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg
Lys Arg145 150 155 160Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr
Leu Ile His Gln Ser 165 170 175Ile Thr Gly Leu Tyr Glu Thr Arg Ile
Asp Leu Ser Gln Leu Gly Gly 180 185 190Asp Ser Pro Lys Lys Lys Arg
Lys Val Glu Ala Ser Gly Gly Gly Gly 195 200 205Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Ala Pro Gly Lys Lys 210 215 220Arg Pro Val
Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr225 230 235
240Gly Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln
245 250 255Thr Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly
Gln Pro 260 265 270Pro Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met
Ala Thr Gly Ser 275 280 285Gly Ala Pro Met Ala Asp Asn Asn Glu Gly
Ala Asp Gly Val Gly Asn 290 295 300Ser Ser Gly Asn Trp His Cys Asp
Ser Thr Trp Met Gly Asp Arg Val305 310 315 320Ile Thr Thr Ser Thr
Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 325 330 335Leu Tyr Lys
Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His 340 345 350Tyr
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 355 360
365His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn
370 375 380Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn
Ile Gln385 390 395 400Val Lys Glu Val Thr Gln Asn Asp Gly Thr Thr
Thr Ile Ala Asn Asn 405 410 415Leu Thr Ser Thr Val Gln Val Phe Thr
Asp Ser Glu Tyr Gln Leu Pro 420 425 430Tyr Val Leu Gly Ser Ala His
Gln Gly Cys Leu Pro Pro Phe Pro Ala 435 440 445Asp Val Phe Met Val
Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 450 455 460Ser Gln Ala
Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro465 470 475
480Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe
485 490 495Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
Leu Asp 500 505 510Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr
Tyr Leu Ser Arg 515 520 525Thr Asn Thr Pro Ser Gly Thr Thr Thr Gln
Ser Arg Leu Gln Phe Ser 530 535 540Gln Ala Gly Ala Ser Asp Ile Arg
Asp Gln Ser Arg Asn Trp Leu Pro545 550 555 560Gly Pro Cys Tyr Arg
Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn 565 570 575Asn Asn Ser
Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn 580 585 590Gly
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys 595 600
605Asp Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly
610 615 620Lys Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val
Met Ile625 630 635 640Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro
Val Ala Thr Glu Gln 645 650 655Tyr Gly Ser Val Ser Thr Asn Leu Gln
Arg Gly Asn Arg Gln Ala Ala 660 665 670Thr Ala Asp Val Asn Thr Gln
Gly Val Leu Pro Gly Met Val Trp Gln 675 680 685Asp Arg Asp Val Tyr
Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 690 695 700Thr Asp Gly
His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu705 710 715
720Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala
725 730 735Asn Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe
Ile Thr 740 745 750Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu
Trp Glu Leu Gln 755 760 765Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu
Ile Gln Tyr Thr Ser Asn 770 775 780Tyr Asn Lys Ser Val Asn Val Asp
Phe Thr Val Asp Thr Asn Gly Val785 790 795 800Tyr Ser Glu Pro Arg
Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 805 810
8159816PRTArtificial SequenceSynthetic Polypeptide 9Met Ile Lys Ile
Ala Thr Arg Lys Tyr Leu Gly Lys Gln Asn Val Tyr1 5 10 15Asp Ile Gly
Val Glu Arg Asp His Asn Phe Ala Leu Lys Asn Gly Phe 20 25 30Ile Ala
Ser Asn Cys Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly 35 40 45Asn
Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala 50 55
60Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys65
70 75 80Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile
Glu 85 90 95Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala
Asn Leu 100 105 110Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp
Lys Pro Ile Arg 115 120 125Glu Gln Ala Glu Asn Ile Ile His Leu Phe
Thr Leu Thr Asn Leu Gly 130 135 140Ala Pro Ala Ala Phe Lys Tyr Phe
Asp Thr Thr Ile Asp Arg Lys Arg145 150 155 160Tyr Thr Ser Thr Lys
Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser 165 170 175Ile Thr Gly
Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly 180 185 190Asp
Ser Pro Lys Lys Lys Arg Lys Val Glu Ala Ser Glu Ala Ala Ala 195 200
205Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Pro Gly Lys Lys
210 215 220Arg Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser
Gly Thr225 230 235 240Gly Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg
Leu Asn Phe Gly Gln 245 250 255Thr Gly Asp Ala Asp Ser Val Pro Asp
Pro Gln Pro Leu Gly Gln Pro 260 265 270Pro Ala Ala Pro Ser Gly Leu
Gly Thr Asn Thr Met Ala Thr Gly Ser 275 280 285Gly Ala Pro Met Ala
Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn 290 295 300Ser Ser Gly
Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val305 310 315
320Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
325 330 335Leu Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp
Asn His 340 345 350Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp
Phe Asn Arg Phe 355 360 365His Cys His Phe Ser Pro Arg Asp Trp Gln
Arg Leu Ile Asn Asn Asn 370 375 380Trp Gly Phe Arg Pro Lys Arg Leu
Asn Phe Lys Leu Phe Asn Ile Gln385 390 395 400Val Lys Glu Val Thr
Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn 405 410 415Leu Thr Ser
Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro 420 425 430Tyr
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 435 440
445Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly
450 455 460Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr
Phe Pro465 470 475 480Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr
Phe Ser Tyr Thr Phe 485 490 495Glu Asp Val Pro Phe His Ser Ser Tyr
Ala His Ser Gln Ser Leu Asp 500 505 510Arg Leu Met Asn Pro
References